Plant Tissue Culture What is plant - PowerPoint Presentation

Slide1

Plant Tissue Culture

Slide2

What is plant tissue culture

?

Plant tissue culture is a technique of growing plant cells, tissues, organs, seeds or other plant parts in a sterile environment on a nutrient medium

Slide3

WHY?

The production of

clones 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.

Slide4

WHY?

The

production of plants in sterile containers reduces disease transmissionAllows production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and 

Nepenthes.To clean particular plants of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture.

Slide5

Terminology

Explant

Living tissue transferred

from a plant to an artificial

medium for culture.

It can be any portion of

the shoot, leaves, roots,

flower or cells from a plant.

Slide6

How?

Adult plant cells are

totipotent, meaning they have the ability to give rise to a fully differentiated plant. Because of this, it is possible to collect cells from a mature plant and use those cells to produce clones of that plant.

Slide7

Plant tissue Culture Basics

Modern plant tissue culture is performed under

aseptic conditionsLiving plant materials from the environment are naturally contaminated on their surfaces (and sometimes interiors) with microorganisms, so surface sterilization of starting material (

explants) in chemical solutions (usually alcohol and sodium or calcium hypochlorite is required).

Slide8

Plant tissue Culture Basics

Explants

are then usually placed on the surface of a solid culture medium, but are sometimes placed directly into a liquid medium, when cell suspension cultures are desired.

Culture media are generally composed of inorganic salts plus a few organic nutrients, vitamins and plant hormones.

Slide9

Plant Tissue Culture Media

Slide10

Salt Mixtures

Organic Substances

Natural Complexes

Inert Supportive Materials

Growth Regulators

Slide11

Macro-nutrient salts

What the ?

NH4NO3 Ammonium nitrate

KNO3 Potassium nitrate

CaCl2 -2 H2O Calcium chloride (Anhydrous)

MgSO4 -7 H2O Magnesium sulfide (Epsom Salts)

KH2PO4 Potassium hypophosphate

FeNaEDTA Fe/Na ethylene-diamine-tetra

acetate

H3BO3 Boric Acid

MnSO4 - 4 H2O Manganese sulfate

ZnSO4 - 7 H2O Zinc sulfate

KI Potassium iodide

Na2MoO4 - 2 H2O Sodium molybdate

CuSO4 - 5 H2O Cupric sulfate

CoCl2 - H2O Cobaltous sulfide

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Macronutrient salts

Nitrogen

– Influences plant growth rate, essential in plant nucleic acids (DNA), proteins, chlorophyll, amino acids, and hormones.

Phosphorus – Abundant in meristematic and fast growing tissue, essential in photosynthesis, respiration.

Potassium

–

Necessary for cell division, meristematic tissue, helps in the pathways for carbohydrate, protein and chlorophyll synthesis.

Slide13

Macronutrient salts

Calcium

-

Involved in formation of cell walls and root and leaf development. Participates in translocation of sugars, amino acids, and ties up oxalic acid (toxin).

Iron

-

Involved in respiration , chlorophyll synthesis and photosynthesis.

FeNaEDTA

=

sodium salt of

EDTA sequesters iron, making it available to plants

.

Magnesium -

Involved in photosynthetic and respiration systems. Active in uptake of phosphate and translocation of phosphate and starches.

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Micronutrient salts

Sulfur

-

Involved in formation of nodules and chlorophyll synthesis, structural component of amino acids and enzymes.

Manganese

-

Involved in regulation of enzymes and growth hormones. Assists in photosynthesis and respiration.

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Micronutrient salts

Molybdenum

-

Involved in enzymatic reduction of nitrates to ammonia. Assists in conversion of inorganic phosphate to organic form.

Zinc -

Involved in production of growth hormones and chlorophyll. Active in respiration and carbohydrate synthesis.

Boron

- Involved in production of growth hormones and chlorophyll. Active in respiration and carbohydrate synthesis.

Copper -

Involved in photosynthetic and respiration systems. Assists chlorophyll synthesis and used as reaction catalyst.

Slide16

Organic Compounds

Carbon Sources – Sucrose, sometimes Glucose or Fructose (Plants Need Carbon)

Vitamins –

Adenine – part of RNA and DNAInositol – part of the B complex, in phosphate form is part of cell membranes, organelles and is not essential to growth but beneficial

Thiamine –

essential as a coenzyme in the citric acid cycle.

Slide17

Still other organics

Organic Acids

Citric acid (150 mg/l) typically used with ascorbic acid (100 mg/l) as an antioxidant.Can also use some of Kreb Cycle acidsPhenolic compoundsPhloroglucinol - Stimulates rooting of shoot sections

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Natural Complexes

Coconut endosperm

Fish emulsionProtein hydrolysatesTomato juiceYeast extracts

Malt extractPotato agar

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Growth regulators

auxin - Roots

cytokinin - Shootsgibberellin – Cell Enlargementabscisic acid – Plant stress hormoneethylene – BAD!

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Auxins

Callus formation, rooting of cuttings, and the induction of adventive embryogenesis

IAA

IBA

NAA

2,4-D

2,4,5-T

Picloram

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Cytokinins

-Enhances adventitious shoot formation

BA 2iP KinetinZeatin

Slide22

Gibberellin

Not generally used in tissue culture

Tends to suppress root formation and adventitious embryo formation

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Abscisic Acid

Primarily a growth inhibitor but enables more normal development of embryos, both zygotic and adventitious

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Ethylene

Question is not how much to add but how to get rid of it

in-vitroNatural substance produced by tissue cultures at fairly high levels especially when cells are under stress

Enhances senescenseSupresses embryogenesis and development in general

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Hormone Combinations

Callus development

Adventitious embryogenesisRooting of shoot cuttingsAdventitious shoot and root formation

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Applications[

edit]

Plant tissue culture is used widely in the plant sciences, forestry, and in horticulture. Applications include:The commercial production of plants used as potting, landscape, and florist subjects, which uses meristem and shoot culture to produce large numbers of identical individuals

.1.To conserve rare or endangered plant species.[6

]

2.A

 

plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicide resistance/tolerance

.

3.Large-scale

growth of plant cells in liquid culture in 

bioreactors

 for production of valuable compounds, like 

plant-derived secondary metabolites

 and 

recombinant proteins

 used as 

biopharmaceuticals

.

[7

]

4.To

cross distantly related species by 

protoplast fusion

 and regeneration of the novel 

hybrid

.

]

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5.To

rapidly study the molecular basis for physiological, biochemical, and reproductive mechanisms in plants, for example in vitro selection for stress tolerant plants.

[8]6.To cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die (Embryo Rescue

).7.For chromosome doubling and induction of polyploidy,

[9]

 for example doubled haploids, 

tetraploids

, and other forms of polyploids. This is usually achieved by application of antimitotic agents such as colchicine

 or 

oryzalin

.

8.As

a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of 

transgenic

 plants

.

9.Certain

techniques such as meristem tip culture can be used to produce clean plant material from

virused

stock, such as sugarcane

[10]

, potatoes and many species of soft fruit.

Production of identical sterile hybrid species can be obtained

.

10.Large

scale production of artificial seeds through

somatic

embryogenesis

[11