• 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; different resultant 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 of genetic make-up of resultant plant identical to that of

the parent and other offspringthe parent and other offspring– Common, allowing them to survive habitats Common, allowing them to survive habitats – Independent of pollinating vectorsIndependent of pollinating vectors

Angiosperm Life Cycle

Natural Cloning

stolon

rhizome

corm

tuber bulb

KalanchoeKalanchoe

Vegetatively – propagated crops

Alternative Propagation Method: Tissue culture

In an effort to increase productivity, alternative propagation methods have been developed.

Plant PropagationPlant Propagation

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

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

Totipotency of Plant CellsTotipotency of Plant Cells

Plant cells possess profound ability to Plant cells possess profound ability to show their full genetic potential and show their full genetic potential and follow a developmental pathway similar follow a developmental pathway similar to that of the zygote resulting in the to that of the zygote resulting in the formation of a new plant.formation of a new plant.

PTCPTC

• Demonstration of totipotency of plant Demonstration of totipotency of plant cellscells

– Ability of the differentiated cell to revert Ability of the differentiated cell to revert to its undifferentiated state and form all to its undifferentiated state and form all parts of a mature organism parts of a mature organism

– Similar to the ability of a zygote to Similar to the ability of a zygote to generate a complete plantgenerate a complete plant

Genomic equivalenceGenomic equivalence Different kinds of somatic cells in Different kinds of somatic cells in

organisms all have the same genesorganisms all have the same genes

Differences between cells in a Differences between cells in a multicellular organism come from multicellular organism come from differences in gene expressiondifferences in gene expression

PTCPTC

DNA

Primary RNA transcript

proteininactive 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

Tissue cultureTissue culture

• Technique for maintaining plant Technique for maintaining plant tissues indefinitely on an artificial tissues indefinitely on an artificial mediummedium

subcultured

Callus

Callus

undifferentiated

roots

redifferentiation shoots

Somatic embryos

organogenesis

Somatic

embryogenesis

subcultured

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

PTCPTC

*For conservation of biodiversity and genetic *For conservation of biodiversity and genetic resourcesresources

*For elimination of some diseases in plants, *For elimination of some diseases in plants, particularly those caused by virusesparticularly those caused by viruses

• Micropropagation- large scale cloning of Micropropagation- large scale cloning of plant speciesplant species– Meristem cultureMeristem culture

•Propagation of rare speciesPropagation of rare species

•Pathogen - free propagulesPathogen - free propagules– Sometimes exhibits somaclonal Sometimes exhibits somaclonal

variationvariation

Uses of biotechnologyUses of biotechnology

• Crop improvementCrop improvement

– Salt toleranceSalt tolerance

– Insect resistanceInsect resistance

– Herbicide resistance Herbicide resistance

•e.g Brassica campestris herbicide resistance e.g Brassica campestris herbicide resistance into Brassica napusinto Brassica napus

Uses of biotechnologyUses of biotechnology

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

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 initiated2. Purpose of the proposed culture3. Plant species to be used

PTCPTC

PTCPTC

SterilizationBiggest 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. 

PTCPTC

Duration of surface sterilization is important:

Too long: plant tissue will be damagedToo 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

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.

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

PTCPTCCulture medium

• The components of a plant tissue culture The components of a plant tissue culture medium include:medium include:– Macronutrients- provide C,N,P,K, Ca, Mg Macronutrients- provide C,N,P,K, Ca, Mg

and Sand S– Micronutrients in trace amounts- Mn, Cu, Micronutrients in trace amounts- Mn, Cu,

Zn, Mo, CoZn, Mo, Co– Iron supplementIron supplement– VitaminsVitamins– Carbon sourceCarbon source– Plant growth regulatorsPlant growth regulators

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

Classes of plant growth substances:

1. Auxins.2. Cytokinins3. Gibberellins4. Ethylene5. Abscissic acid6. Brassinosteroids

Auxins and cytokinins are the most important for regulating growth and morphogenesis in plant tissue culture

PTCPTC

Auxin/cytokinin interactionAuxin/cytokinin interaction

auxinhigh

low

cytokinin

high

lowRoot formation from

shoot

Adventitious root formation from callus

Callus induction in dicotyledons

Adventitious shoot formation

Axillary shoot proliferation

Fern spore germination in a plant tissue culture system

Lilian B. Ungson, Ph.D Professorial Lecturer

Institute of Biology

U. P. Diliman, Quezon City

Fern Life Cycle

Platycerium

Asplenium musifoliumAsplenium musifolium

Adiantum sp.(Maiden Hair Fern)

Adiantum

Cyathea contaminans(Tree Fern)

Cyathea contaminans(Tree Fern)

Cyathea contaminans(Tree Fern)

Christella

Christella

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

•ls

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.

•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

The fern gametophyte stageshas its origin in a single cell like the carrot cell culture.

Comparisons madebetween molecular changesassociated with differentiationof the carrot cell and germi-nation of the fern spore andform changes in the fern gametophyte are germane(Raghavan 1993).

Germination of pollen and germination of fern sporesboth involve activation of growth and induction of metabolicactivities in dormant systems

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

Annulus=thick-walledplate or transverseband or ring that extendaround thecircumference of the sporangium

Function in spore release

Spore patch

Platycerium coronarium

Spores inside the sporangiumSpores inside the sporangium

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.

Cytological changes during maturation of spore Onoclea

a. Beginning of vacuolation. during spore enlargementb. Nucleus at one end of

sporec. Spore enlargement

accompanied by decrease in cytoplasm

d. Enlargement of nucleus prior to dev of proplastids. e. First appearance of

proplastids around nucleusf. Continued dev of

proplastidsg. Mature spore with central nucleus and numerous

chloroplasts

SPORE- represents the beginning of the haploid or gametophytic phase

Nucleus with dehydratedchromatin

Dormant spore

Storage granules

Storage granules have tobe degraded into simplecompounds to provideenergy and substratesfor germinationNeed for synthesis ofnucleic acids Need for biogenesis of organelles e.g. mitochondria for catabolic activity of food reserves and chloroplasts for initiating photosynthesisHistochemical observation: most storage granules disappear within 24 to 36 hours after germination

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)

Germination of spores

nucleus

Cell wallformation delimitingrhizoid initial

rhizoid initial Elongated rhizoid

Protonemainitial

A

B

C

C rhizoid breaking out of exine

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

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

Germinated spore of Blechnum spicant to show relationship between rhizoid and protonema initial

Protonema initial

Rhizoid initial

nucleus

Whole mounts of germinating spores of Onoclea sensibilis

A. Asymmetric division delimiting the rhizoid.B. Elongation of the rhizoidC.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

Rhizoid initial growth is tip growthSimilar to pollen tube

it is a cell that grows by apical extension like pollen tubesand root hair

programmed for terminal differentiation, does not normally divide after it is cut off

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

Pollen tube

Rhizoid

Protonemal initial

Ungerminated spore

Rhizoid initial

Filamentous protonema

Spore Germination

Gives riseto green leafy gametophyte

Programmedfor terminaldifferentiation

Rhizoid initial

Nucleus is confined to the base of a newly-cutrhizoid initialSurrounded by a chloro- plasts, mitochondria, ribo-Somes, Golgi, ER, vesicles,

Extensive vacuolation- associated with elongationChloroplasts degenerate , lose integrity of their thylakoid membraneEscape of starch grains.

Protonemal initial

Progenitor of the prothallusDistinctive 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.

Formation of protonema

A

B

C

A. early stage, formalmost identicalcells

B. Division in both cells to form filaments

C. Initiation of planar growth in both filaments

A B

Rhizoid initiation in the presence of actinomycin DRhizoid initiation

Basal medium without actinomycin DNormal germination

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

Start of Planar Morphology

Prothallial Development

Filamentous growth: Protonemal initial formed by division of transverse walls

Planar morphology: by rapidburst of transverse and longitudinal divisions

Rhizoid-programmedfor terminal differentiation

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

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

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 cellApical cell divides transversely

Germination of fern spores and development of gametophyte

Rhizoid develops from basal cellProthallial cell may divide transversely several times to form a filamentPlate of cells is formed by longitudinal divisionsGrowth becomes active along forward margins of the thallus which results in formation of two wings.

Assuming a heart-shaped structure – Prothallus

(Fern Gametophyte)

Prothallial Development

Mature ProthallusMature Prothallus

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

Sex organs on gametophytes

Development of antheridium (A,B) and archegonium (C-H)

Primary spermatogenous orandrogonial cells divide several times to form androcytes then transformed into spermatozoids

Mature antheridium with sperms forming in spermatogenous cells

Mature sperm – large, coiled and ciliated

A. Archegonial initial divided intoinner and outer cell and outer celldivided 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 celland neck canal cell

D. Division of neck canal cell

E. Nearly mature archegonium withegg, ventral canal cell and binucleate neck canal cell

F. Archegonium with egg ready for fertilization

E

 

 

Day 7Rhizoid and protonema

Day 9Rhizoid and protonema

Day 14 Young prothallus

Day 49 Sporophyte on gametophyte

Day 42Development ofantheridia

Fern culture Day 65

Sporophytes on drying gametophyte

Sporophytes on gametophytes

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

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.