2. • Sexual CycleSexual Cycle
– new plants arise from the fusion of parental gametesnew plants arise from the fusion of parental gametes
– development from seedsdevelopment from seeds
– plants propagated by seeds are not clonesplants propagated by seeds are not clones
– resultant plant has unique genetic make-up; differentresultant plant has unique genetic make-up; different
from either parent and other offspringfrom either parent and other offspring
• Asexual or Vegetative CycleAsexual or Vegetative Cycle
– genes copied exactly at each mitotic divisiongenes copied exactly at each mitotic division
– genetic make-up of resultant plant identical to that ofgenetic make-up of resultant plant identical to that of
the parent and other offspringthe parent and other offspring
– Common, allowing them to survive habitatsCommon, allowing them to survive habitats
– Independent of pollinating vectorsIndependent of pollinating vectors

3. Angiosperm Life Cycle

4. Natural Cloning
stolon
rhizome
corm
tuber bulb

5. KalanchoeKalanchoe

6. Vegetatively – propagated
crops

7. Alternative Propagation Method: Tissue culture
In an effort to increase productivity, alternative
propagation methods have been developed.
Plant PropagationPlant Propagation

8. Vegetative propagation of importance to agriculture,
horticulture and forestry since it provides:
1.For the production of uniform material
for crop planting,
2.For the multiplication of good quality or
superior trees, ornamentals, vegetables etc.
Plant PropagationPlant Propagation

9. The term “tissue culture”
is an inclusive name for both
organ and cell culture.
Plant tissue culture
 the growing of isolated plant parts aseptically, on
appropriate media and a whole new plant can be
produced.
Utilizes growth of small pieces of tissue or small
organs in sterile or aseptic conditions
“in vitro” techniques ((literally means “in a glass”
PTCPTC

10. Totipotency of Plant CellsTotipotency of Plant Cells
Plant cells possess profound ability to showPlant cells possess profound ability to show
their full genetic potential and follow atheir full genetic potential and follow a
developmental pathway similar to that of thedevelopmental pathway similar to that of the
zygote resulting in the formation of a newzygote resulting in the formation of a new
plant.plant.

11. PTCPTC
• Demonstration of totipotency of plant cellsDemonstration of totipotency of plant cells
– Ability of the differentiated cell to revert to itsAbility of the differentiated cell to revert to its
undifferentiated state and form all parts of aundifferentiated state and form all parts of a
mature organismmature organism
– Similar to the ability of a zygote to generate aSimilar to the ability of a zygote to generate a
complete plantcomplete plant

12.  Genomic equivalenceGenomic equivalence
 Different kinds of somatic cells inDifferent kinds of somatic cells in
organisms all have the same genesorganisms all have the same genes
 Differences between cells in a multicellularDifferences between cells in a multicellular
organism come from differences in geneorganism come from differences in gene
expressionexpression
PTCPTC

13. DNA
Primary RNA
transcript
protein
inactive mRNA
Inactive protein
mRNA degradation
control
Translational control
by ribosome selection
among mRNAs
Protein activity
control
Transcriptional
control
1
2 Processing
control
3 Transport
control
mRNA
mRNA
6
4 5
Steps at which
gene expression
can be controlled
in eukaryotes
NUCLEUS
CYTOPLASM

14. Tissue cultureTissue culture
• Technique for maintaining plant tissuesTechnique for maintaining plant tissues
indefinitely on an artificial mediumindefinitely on an artificial medium
subcultured
Callus
Callus
undifferentiated
roots
redifferentiation
shoots
Somatic
embryos
organogenesis
Somatic
embryogenesis
subcultured

16.  For rapid vegetative propagation of plants
 For production and extraction of valuable
secondary metabolites rather than directly
from plants grown in the wild
APPLICATIONS
PTCPTC

17. PTCPTC
*For conservation of biodiversity and genetic resources*For conservation of biodiversity and genetic resources
*For elimination of some diseases in plants, particularly*For elimination of some diseases in plants, particularly
those caused by virusesthose caused by viruses

18. • Micropropagation- large scale cloning of plantMicropropagation- large scale cloning of plant
speciesspecies
– Meristem cultureMeristem culture
• Propagation of rare speciesPropagation of rare species
• Pathogen - free propagulesPathogen - free propagules
– Sometimes exhibits somaclonal variationSometimes exhibits somaclonal variation
Uses of biotechnologyUses of biotechnology

19. • Crop improvementCrop improvement
– Salt toleranceSalt tolerance
– Insect resistanceInsect resistance
– Herbicide resistanceHerbicide resistance
• e.g Brassica campestris herbicide resistance intoe.g Brassica campestris herbicide resistance into
Brassica napusBrassica napus
Uses of biotechnologyUses of biotechnology

20. Two major underlying principles
1. The necessity to isolate the
plant part from the intact plants
2. The need to provide the appropriate environment
in which the isolated plant part can express its
intrinsic or induced potential through the use of a
suitable culture media and their proper culture
conditions.
Initiating Tissue Culture
PTCPTC

21. Explant and Explant Sources
Pieces of whole plants, small organ itself or pieces of
tissue from stems, leaves, ovules, seeds, buds,
inflorescence. The part of the plant from which
explants are obtained depends on :
1. Type of culture to be initiated
2. Purpose of the proposed culture
3. Plant species to be used
PTCPTC

22. PTCPTC
Sterilization
Biggest preoccupation of a plant tissue culturist is how
to prevent contamination of the culture. Presence of
microorganisms in cultures results in loss of time,
energy and money.

23. PTCPTC
Duration of surface sterilization is important:
Too long: plant tissue will be damaged
Too short: will not destroy the microorganisms
Usually: ca. 20 minutes in 5% calcium hypochlorite
and 5-15 minutes in 0.5- 1.0 % sodium
hypochlorite

24. PTCPTC
Cultural Factors
Sterile operations are conducted within a
laminar flow cabinet. With a laminar flow
cabinet air taken from outside of the
hood is forced through a dust filter and
then the filtered air which passed
through a high efficiency particulate air
(HEPA) filter is blown in a very smooth
laminar flow towards the user or out of
the workplace.
The filters can remove up to 99.97% of
dust, pollen, molds, bacteria and other
airborne particles as small as 0.3
microns.

25. Cultural Factors
Explants are put into a sterilized nutrient medium.
It is absolutely necessary to maintain a sterile
environment during the culture of plant tissues.
PTCPTC

26. PTCPTC
Culture medium
• The components of a plant tissue culture mediumThe components of a plant tissue culture medium
include:include:
– Macronutrients- provide C,N,P,K, Ca, Mg and SMacronutrients- provide C,N,P,K, Ca, Mg and S
– Micronutrients in trace amounts- Mn, Cu, Zn,Micronutrients in trace amounts- Mn, Cu, Zn,
Mo, CoMo, Co
– Iron supplementIron supplement
– VitaminsVitamins
– Carbon sourceCarbon source
– Plant growth regulatorsPlant growth regulators

27. Plant growth regulators
The growth regulator requirements for most callus cultures
are some combinations of auxins and cytokinins. They are
organic substances which are active at very low
concentrations (10-5
to 10-9
M), can elicit profound cellular
changes influencing plant development.
PTCPTC

28. Classes of plant growth substances:
1. Auxins.
2. Cytokinins
3. Gibberellins
4. Ethylene
5. Abscissic acid
6. Brassinosteroids
Auxins and cytokinins are the most important for
regulating growth and morphogenesis in plant tissue
culture
PTCPTC

29. Auxin/cytokinin interactionAuxin/cytokinin interaction
auxin
high
low
cytokinin
high
low
Root formation from
shoot
Adventitious root formation from
callus
Callus induction in dicotyledons
Adventitious shoot formation
Axillary shoot proliferation

30. Fern spore germination in a
plant tissue culture system
Lilian B. Ungson, Ph.D
Professorial Lecturer
Institute of Biology
U. P. Diliman, Quezon City

31. Fern Life Cycle

32. Platycerium

33. Asplenium musifoliumAsplenium musifolium

34. Adiantum sp.
(Maiden Hair Fern)
Adiantum

35. Cyathea contaminans
(Tree Fern)

36. Cyathea contaminans
(Tree Fern)

37. Cyathea contaminans
(Tree Fern)

38. Christella

40. Christella

41. Fern for experimental studies
• Ease of culture of using gametophytes
at different stages of development
•Spores and gametophytes are small, can be
cultured in petri dishes
•Large populations can produce data which
can be subjected to statistical analysis
•Advantages derived from the intrinsic features of
fern life cycle
• Single-celled nature of the spore
• Spore germinates to form cells destined for different fates
• Growth of gametophytes as a single layer of cells
for study of cell division patterns
• Growth of the filamentous structure
• Formation of sex organs in response to hormonal signals

42. Spore culture in a plant tissue culture system
Raghavan in 1993 said that fern haplophase is not considered
as a tissue culture system in the accepted sense
of the terminology (Raghavan 1993): plant tissue
culture is a collection of techniques used to grow
explants on formulated media for induction of growth,
differentiation, and regeneration of organs or
whole organisms.

43. •Cells dedifferentiate in
tissue culture and can give
rise to the diverse cell types,
thus it possess all the genes
necessary to make any kind
of plant cell.
Carrot cell culture

44. The fern gametophyte stages
has its origin in a single cell like
the carrot cell culture.
Comparisons made
between molecular changes
associated with differentiation
of the carrot cell and germi-
nation of the fern spore and
form changes in the fern
gametophyte are germane
(Raghavan 1993).
Germination of pollen and
germination of fern spores
both involve activation of growth
and induction of metabolic
activities in dormant systems

45. Each sorus has a
•central axis to which
the sporangia are
attached;
• Indusium-covering
underneath the
sporangia,
• Annulus –thick-walled
ring of cells around
each sporangium .The
annulus is hygroscopic
Fern leaf with sorus
indusiumAnnuluS

46. Annulus=thick-walled
plate or transverse
band or ring that extend
around the
circumference
of the sporangium
Function in
spore release

47. Spore patch

48. Platycerium coronarium

49. Spores inside the sporangiumSpores inside the sporangium

50. Internal condition of the spore
• Freshly released spores are immature
• Need to complete series of cytological
and biochemical changes for maturation
and germination
• Cytological changes ensure formation
and orderly rearrangement of organelles
• Maturation is due to the endogenous synthesis
of a variety of molecules.

51. Cytological changes during maturation of spore Onoclea
a. Beginning of vacuolation.
during spore enlargement
b. Nucleus at one end of
spore
c. Spore enlargement
accompanied by decrease
in cytoplasm
d. Enlargement of nucleus
prior to dev of proplastids.
e. First appearance of
proplastids around nucleus
f. Continued dev of
proplastids
g. Mature spore with central
nucleus and numerous
chloroplasts

52. SPORE- represents the beginning of the haploid or
gametophytic phase
Nucleus with dehydrated
chromatin
Dormant spore
Storage
granules
Storage granules have to
be degraded into simple
compounds to provide
energy and substrates
for germination
Need for synthesis of
nucleic acids
Need for biogenesis of organelles
e.g. mitochondria for catabolic activity of food reserves
and chloroplasts for initiating photosynthesis
Histochemical observation: most storage granules disappear
within 24 to 36 hours after germination

53. Chloroplast movement and
origin of polarity in germinating
spores
a. Unpolarized spore
showing uniform
distribution of
chloroplast around
nucleus.
b. Beginning of
polarized move-
ment of chloroplasts
away from
site of presumptive
rhizoid initial.
c. Spore nucleus in
mitosis to form the
rhizoid initial
d. Formation of the
rhizoid initial
( arrow)

54. Germination of spores
nucleus
Cell wall
formation delimiting
rhizoid initial
rhizoid initial Elongated rhizoid
Protonema
initial
A
B
C
C rhizoid breaking out of exine

55. An asymmetric cell division is the cytological hallmark of
germination of fern spores
The development potential of the spore is parceled out to
2 cells which pursue divergent differentiation pathways.
rhizoid initial-- small & lens-shaped, elongates into
1. narrow colorless rhizoid.
2. large cell divides again by a wall perpendicular to
the first to form an isodiametric cell called
protonema initial with many chloroplasts
Appearing tip of the nascent rhizoid initial –first visible sign of
germination
Spore germination

56. Section of germinated spore of Polypodium vulgare
Protonema initial
Rhizoid initial
The basal wall of
the rhizoid is in
contact with the
protonema initial
and the spore cell
Spore cell with a
large mass of
lipid bodies

57. Germinated spore of Blechnum spicant to show
relationship between rhizoid and protonema initial
Protonema initial
Rhizoid initial
nucleus

58. Whole mounts of germinating spores of Onoclea sensibilis
A. Asymmetric division delimiting the rhizoid.
B. Elongation of the rhizoid
C.Formation of the protonemal initial
Germination of the spore -a process of change from a dormant
unicell to a pair of morphologically and functionally different cells
A

59. Rhizoid initial growth is tip growth
Similar to pollen tube
 it is a cell that grows by apical extension like pollen tubes
and root hair
 programmed for terminal differentiation,
 does not normally divide after it is cut off

60. A. Tip domain- rich in Golgi vesicles
B. Sub-apical domain- with metabolically active organelles:
mitochondria, dictyosomes, ER, vesicles
C. Nuclear domain: large organelles and male germ unit
D. Vacuolar domain. Enlarges as the tube grows.
Pollen tube

61. Rhizoid
Protonemal initial
Ungerminated spore
Rhizoid initial
Filamentous protonema
Spore Germination
Gives rise
to green leafy
gametophyte
Programmed
for terminal
differentiation

62. Rhizoid initial
Nucleus is confined to the
base of a newly-cut
rhizoid initial
Surrounded by a chloro-
plasts, mitochondria, ribo-
Somes, Golgi, ER, vesicles,
Extensive vacuolation-
associated with elongation
Chloroplasts degenerate ,
lose integrity of their
thylakoid membrane
Escape of starch grains.

63. Protonemal initial
Progenitor of the prothallus
Distinctive feature is abundant
chloroplasts
 Cytoplasm is filled with much lipid
bodies, protein granules and
chloroplasts.
Divides by walls perpendicular
to the long axis to produce a
filament.

64. Formation of protonema
A
B
C
A. early stage,
form
almost identical
cells
B. Division in
both cells to form
filaments
C. Initiation of
planar growth in
both filaments

65. A B
Rhizoid initiation in the
presence of actinomycin D
Rhizoid initiation
Basal medium without
actinomycin D
Normal germination

66. Stored mRNA
In seeds and fungal spores, there is stored template
mRNA carry codes for the first proteins of germination
Hypothesis: fern spores also contain stored mRNA.
Believed that sufficient mRNA translatable into proteins is
stored in the spore as a holdover from sporogenesis
To test hypothesis: use of antibiotic actinomycin D
(known to inhibit mRNA synthesis)
Ground rule established: if a stage of germination
proceeded in the presence of actinomycin D in the
medium, the event was probably independent of
synthesis of NEW mRNA

67. Start of Planar
Morphology
Prothallial Development
Filamentous growth: Protonemal initial
formed by division of transverse walls
Planar morphology: by rapid
burst of transverse and
longitudinal
divisions
Rhizoid-programmed
for terminal
differentiation

68. Gametophytes of Asplenium showing longitudinal
division of the terminal cells
Formation of the planar
gametophyte due to activity of
single terminal cells.
Ist div is oblique or longitu-
dinal
Followed by partition at right
angles to the first producing
a group of three cells. Center
cell (wedge-shaped) functions
as the meristematic apical cell

69. Planar growth in producing a prothallus
a. Filamentous protonema, b. ist longitudinal div of terminal cell
c. Formation of a wall at right angles to the first division wall
d. Spatulate plate is formed by repeated oblique or longitudinal
divisions with left-right alternation of cell plate orientation

70. Prothallus development- how heart-shape is attained
 At first apical cell appears as a small indentation at tip of
spatulate plate. (d, e)
 Later, the two sides extend horizontally assuming a heart-
shape form and meristematic cell is lodged in the notch
between the two lobes.(e, f, g)
 During further expansion of the lobes, the apical cell divides
transversely (f,g,)
 Division of anterior cell by two or three cell walls parallel to
each other (g,H)
Meristematic cell
Apical cell divides
transversely

71. Germination of fern
spores and development
of gametophyte
Rhizoid develops from basal
cell
Prothallial cell may divide
transversely several times to
form a filament
Plate of cells is formed by
longitudinal divisions
Growth becomes active
along forward margins of
the thallus which results in
formation of two wings.

72. Assuming a heart-shaped structure – Prothallus
(Fern Gametophyte)
Prothallial Development

73. Mature ProthallusMature Prothallus

74. Sex organs on gametophytes
 When antheridial development precede archegonia,
antheridia are confined to ventral surface behind the
apical notch of the prothallus
Antheridia may be scattered over the entire prothallial
surface or confined to the margins of the prothallus
 When both sex organs develop at the same time, there is
competition for space , nutrients and other resources.
Antheridia are confined to the midrib region in the posterior
half and archegonia to the anterior region of the midrib

75. Sex organs on gametophytes

77. Development of antheridium (A,B) and archegonium (C-H)
Primary
spermatogenous
or
androgonial cells
divide several
times to form
androcytes then
transformed into
spermatozoids

78. Mature antheridium with
sperms forming in
spermatogenous cells
Mature sperm – large,
coiled and ciliated

79. A. Archegonial initial divided into
inner and outer cell and outer cell
divided anticl to form 2 neck cells
B. Formation of central cell and basal
cell from the inner cell.
C. Div of central cell into ventral cell
and neck canal cell
D. Division of neck canal cell
E. Nearly mature archegonium with
egg, ventral canal cell and binucleate
neck canal cell
F. Archegonium with egg ready for
fertilization
E

81. Day 7
Rhizoid and protonema

82. Day 9
Rhizoid and protonema

83. Day 14 Young prothallus

84. Day 49
Sporophyte on gametophyte

85. Day 42
Development of
antheridia

86. Fern culture Day 65
Sporophytes on drying gametophyte

87. Sporophytes on gametophytes

88. Areas for further investigation .
1. Response of spores to light quality and chemicals has many
similarities with behaviour of seeds.
2. Distinct regulatory processes in fern spore germination may
give evidences of additional control mechanisms that are
already established during seed germination.
3. To find out the mechanism of development in a dormant
system at the cellular level of initiation
4. During the development of spores, they require significant
amounts of proteins for storage, and for surviving adverse
conditions. What strategy do spores use to produce these
required amounts of proteins which are in large quantities.
5. What is the trigger that will turn prothallial cells into
archegonia or antheridia

89. References
 Raghavan, V. 1989. Developmental Biology of Fern
Gametophytes. Cambridge University Press, Cambridge.
 Raghavan, V. Cellular and molecular biology of fern
haplophase development. In:Komamine, A., H.
Fukuda, U. Sankawa, Y. Komeda and K. Syono. 1993.
Cellular and Molecular Biology in Plant Cell Cultures. Journal
of Plant Research Special Issue No. 3. The Botanical
Society of Japan, Tokyo
 Reece, R. , L. Urry, M. Cain, S. Wasserman, P. Minorsky,
and R. Jackson. 2011. Campbell Biology.