PLANT BIOTECNOLOGY

REFERENCE BOOKMethods In Plant Tissue Culture -U Kumar (second edition)

-BYSWATI VAISHMBT-10044

TOPICS TO BE COVERED

• Cell Theory • Plant Tissue Culture Techniques• Callus Tissue & Organogenesis• Principles Of Growth• Plant Regeneration• Concept Of Totipotency Of Cells• Plant Tissue Culture Lab• Culture Media• Media Components• Aseptic Techniques• Sterilization Techniques• Points To Remember

Cell Theory

• Cell theory states that:• All living things or

organisms are made of cells & their products.

• New cells are created by old cells dividing into two.

• Cells are basic building units of life.

Plant tissue culture techniques

Callus tissue & organogenesis

Callus on a wounded plant parts or on a culture medium is made up of an amorphous , aggregate of loose parenchyma cells which proliferate from mother cells.

• Callus Formation Is Found On Angiosperms, Gymnosperms, pteridophytes & Bryophytes

• Callus Contains No Organized Meristem

• Callus Is Somewhat Abnormal Tissue which Has Potentiality To Produce Normal Roots & Embroids In Turn It Develops Into Plantlets.

• Callus May Be Hard due To Lignifications Of Cell Walls Or Brittle And Sometimes Soft.

• Organogenesis is the development adventitious organs or primordia (embroid) from undifferentiated cell mass (callus)in tissue culture.

Principles of growth

Growth• Self –multiplication of living materials, the protoplasm itself.

• Increase in size (volume/length) due to cell division and subsequent enlargement.

• Increase in dry weight.

Development• Defined as an ordered change or progress, often towards a higher, more ordered or more complex state.

why growth occurs?Expressed as division of a cell to form two cells and the enlargement of newly divided cells.

Growth kinetics

• First rapid phase.• Maximum growth rate

phase.• Last phase.

Power of regeneration of plants

Concept of totipotency• As cell divide mitotically, they do so

eqautionally to produce daughters cells.

• G.Haberlandt’s claimed that one day it could be possible to rear plants from isolated would be rarely surviving cells of flowering plants.

• He also sated that out of surviving somatic cells artificial embryos would be reared asexually

• Therefore every cell within the plant has a potential to regenerate into a whole plant.

Plant tissue culture lab

• Media preparation room• Culture media, washing

powder/liquid disinfectants.• Aseptic transfer chamber area• Environmentally controlled

culture room• Analytical room• Acclimatization room• Miscellaneous items ( air

conditioner, marker, match box, burner, etc.)

Lab instruments

• pH meter• Balances• Electronic hot air over• Microscopes• Centrifuge• Filter sterilizing equipment• Laminar air flow (LAF)

cabinet.

CULTURE MEDIA

• A Nutrient media generally contains inorganic salts, vitamins, growth regulators, a carbon source & gelling agent.

• Others include –organic nitrogen, hexitols, amino acids, antibiotics & plant extracts.

• Nutrition of callus:• Chemical factors: minimal & plant growth regulators• Environmental factors: light temperature, humidity & genetic

constitution or genotype of the plant.

Media componentsA) INORGANIC SALTS MAJOR ELEMENTS

MAJOR ELEMENTS PROPERTIES DEFICIENCY

Calcium (Ca) •Integral compound of plant cell wall.•Formation of pectin•Promotes root development.•Assists in growth and development.

Iron (Fe) •Chlorophyll synthesis.•Participates in energy conversion in photosynthesis & respiration.

Chorosis (yellowing of leaves)

Magnesium (Mg) •Central element of chlorophyll molecules•Enzyme activator.

Causes older leaves to become chlorotic

Major element properties deficiency

Nitrogen (N) •Influences rate of plant growth rate.•Molecular make of DNA/RNA, Proteins, chlorophyll, amino acids, alkaloids & plant hormones.

Chlorotic leaves & stunted growth

Phosphorus (P) •Meristamatic And Fast Growing Tissue.•Part of DNA & ATP molecule.•Essential in photosynthesis & respiration.

Stunted growth & reddish to purple coloring.

Potassium (K) •Normal cell division•Promotes meristamatic growth

Weak curled or dead leaves

Sulphur (S) •Promotes root development .•Deep green foliage

Yellowing of leaves

MINOR ELEMENTS

MINOR ELEMENTS PROPERTIES DEFICIENCY

Boron (B) •Movement o sugars & waters.•Nitrogen metabolism.•Fruiting &cell division

(less)Heart rot in sugar beet, (more) plant injury or death.

Chlorine (Cl) •Promotes photosynthesis Wilted leaves

Copper (Cu) •Energy conversion.•Chlorophyll synthesis

Stunted growth, malformation twisted leaves

Iodine (I) •Often added as potassium iodide (KI)

Manganese (Mg) •Promotes growth Yellowing of leaves

Molybedenum (Mo)

•Converts nitrogen ammonia and aids nitrogen fixation

Yellowing leaves & stunted growth

Zinc (Zn) • enzyme activator •chlorophyll formation production of IAA

Abnormal rootsDiscoloration of leaf

B) ORGANIC COMPOUNDSCarbohydrates •Sucrose

•D-Mannitol•D-Sorbitol •Hexitols

Vitamins •Vitamin B-complex•Adenine •D-biotin •Choline•Cyanocobalmin•Folic acid•Inositol•Nicotinic acid •PABA•D-Panthothenic acid•Pyridoxine•Riboflavin

C) GROWTH REGULATORSAUXIN • IAA (Indole acetic acid)

•2,4-D•Induction of cell division & root initiation•Callus induction•Stable form

Cytokinins •Zeatin , KN, BAP,2iP•adenine deravatives•Promotes cell division •Shoot proliferation•Organogenesis•Somatic embryo genesis•Differentaition & micropropogation•thermostable

Gibberellins • infrequently used•Inhibits callus growth•But for meristem culture after shoot primordia formation are used in plant regeneration & elongation•Filter sterilized

Abscisic acid •Used in embryo culture & somatic embryo genesis•Heat stable but light sensitive

D) GELLING AGENTS :

•Purified Agar•Difcobacto agar

E) AMINO ACIDS :

•Morphogenesis •L-forms are used•L-tyrosine (shoot initiation)•L-arginine (rooting)•L-serine ( haploid embryogenesis )•L-cysteine ( controls phenol leaching )•L-glutamide (induce somatic embryogenesis)

F) ANTIBIOTCS :•Fungicides •Bactericides•Made fresh & added after autoclaving media

G ) NATURAL COMPLEXES :•Coconut milk (CM)•Yeast extract (YI)•Malt extract (ME)•Tomato juice •Potato extract•casein hydrosylate •Fish emulsion

H) ANTIOXIDANTS :• citric acids •Ascorbic acids •To reduce excessive browning of explants •Adsorbents like PVP & activated charcoal are also used checking excessive browning.

I) ADDITIONAL REQUIREMENTS :• Water quality• Natural & chemical complexes.

Aseptic techniques• Sterilization : destruction of

living matter• Disinfectant : chemical agent

used to kill pathogens without sterilizing matter to which chemical is applied

• Sanitation: substantially reducing & then maintaining the micro-organism population in air & on objects in lab to acceptable levels.

Sterilization of plant tissues

• Sodium hypochloride (NaOCl) : 0.025%-0.25%• Calcium hypochloride (CaOCl): • Hydrogen peroxide (H2O2): 3% -10 %• Bromium water: 1% - 2%• Silver nitrate (AgNO3) : 1%• Mercuric chloride (MgCl2): 0.1 % - 1.1 %

Sterilization Physical

Heat Moist heat : 121oC (10-30 min)

Dry heat : 160oC (1 hour)

Filter 0.75 micrometerMicro filters

Sintered glass filtersAsbestos paper filters

Cellulose membrane filters

Air sterilizationLaminar air flowWith HEPA filters

Irradiation Ultra violet (UV)260 nmHarmful

Acts by inducing thymine-thymine dimmers in DNA .

Chemical Strong disinfectantEg: formaldehyde, ethylene oxide

Mild – ethyl alcoholIodine soap

Mode of action:Acts by denaturing or altering proteins or lipids in the

cytoplasmic membrane of bacterial cell wall.Eg: interfering with energy yielding system in cell :formation

of ATP.

Points to remember

• Cell theory aspects describing the fundamentals of the “CELL”

• CALLUS tissue• Totipotency • Growth & development • Media components• Sterilization technique

Thank you