plant tissue culture ppt

PLANT TISSUE CULTURE

Ph. Islam Adel

Abdelhakim

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Definition: • Plant tissue culture is the science of growing plant

cells, tissues or organs isolated from the Mother plant,

on artificial media in vitro under controlled conditions.

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Edwin F. George, M. A. H., Geert-Jan De Klerk (2008). "Plant Propagation by Tissue Culture

3rd Edition." Volume 1. The Background.

History • HABERLANDT is considered to be the father of plant tissue

culture who conceived the concept of totipotency & cell culture

in 1902.

• Totipotency is the ability of a single cell to divide

and produce all of the differentiated cells in an

organism.

• He was the first to consider culturing cells aseptically in a

nutrient solution.

• However, he was unsuccesful in his culture as he failed to

recognize that cell differentiation requires plant growth

regulators

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Krikorian, A. and D. L. Berquam (1969). "Plant cell and tissue cultures: the role of

Haberlandt." The Botanical Review 35(1): 59-67.

Types of Growth: Organized Growth:

• It occurs when plant cell or tissue (explant) are

transferred to culture media and continue to grow with

their structure preserved.

Unorganized Growth:

• It occurs when pieces of whole plants are cultured in

vitro and the cells aggregate and typically lack any

recognizable structure.

e.g. Callus and Cell Suspension cultures.

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Organized growth Unorganized growth “Callus”

Unorganized growth “Suspension”

Establishment of tissue culture systems

• Explant Selection:

May be from root, stem, leaves or buds.

• Isolation & Sterilization:

Prevention of contamination of

tissue culture media is important

for the whole process of plant

Propagation. So, all the work should

Be performed in special rooms or

Inside hoods or cabinets from which

Microorganisms are excluded.

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Suspension Culture System

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Explant selection and surface sterilization

Callus culture induction on solid media supplemented with sucrose, hormones and

agar

Callus is introduced to agitated liquid media

Gaosheng, H. and J. Jingming (2012). Production of useful secondary metabolites

through regulation of biosynthetic pathway in cell and tissue suspension culture of

medicinal plants, INTECH Open Access Publisher.

Hairy root culture • It is the culture produced after infection of explant and

culture by the gram negative soil bacterium Agrobacterium

rhizogenes.

• This process leads to formation of naturally occuring hairy

root disease.

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Shanks, J. V. and J. Morgan (1999). "Plant ‘hairy root’culture." Current Opinion in

Biotechnology 10(2): 151-155.

The process…

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Plant cell Agrobactrium cell

Induction of hairy root culture

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Explants are wounded and inoculated with A. rhizogenes

2-3 days later, explant transferred to a solid media

with antibiotic as Cefotaxim or Vancomycin.

Hairy roots will be induced within 1-4 weeks

depending on the plant species

Hairy roots separated and cultured on solid media until enough biomass is obtained




Advantages of hairy root cultures:

• The hairy root system is genetically and

biosynthetically stable.

• High production of secondary metabolites.

• The culture can grow under phyto-hormone free

conditions.

• The culture shows fast growth which reduces the

culture time and easy the handling.

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Shanks, J. V. and J. Morgan (1999). "Plant ‘hairy root’culture." Current Opinion in

Biotechnology 10(2): 151-155.

• Growth and development of plant cultures usually

also depends on the addition of plant growth

regulators to the medium.

• They are important in plant tissue culture since they

play vital roles in stem elongation, tropism, and

apical dominance.

• They are generally classified into the following

groups; auxins, cytokinins, gibberellins and abscisic

acid.

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Plant growth regulators

Skoog, F. and C. Miller (1957). Chemical regularion of growth and organ formation in plant

fissue cultured, In vitro. Symp. Soc. Exp. Biol., v. 11, p. 118-131.

• Moreover, proportion of auxins to cytokinins

determines the type and extent of organogenesis in

plant cell cultures.

• ↑ Auxin ↓ Cytokinin = Root Development

• ↑ Cytokinin ↓ Auxin = Shoot Development

• Auxin = Cytokinin = Callus Development

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Zeatin- natural cytokinin

Skoog, F. and C. Miller (1957). Chemical regularion of growth and organ formation in plant

fissue cultured, In vitro. Symp. Soc. Exp. Biol., v. 11, p. 118-131.

In Vitro Cultures and Production of Important Secondary Metabolites

• Secondary metabolites play important role in defencing

insects, herbivores, microbial pathogens, and facilitating

pollination and reproduction.

• Based on the structures, the secondary metabolites can be

classified into alkaloids, flavonoids, phenylpropanoids,

terpenoids, steroids, tannins and proteins.

• These compounds are biosynthesized through series

enzyme catalyzed reactions using simple building blocks in

different ways.

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The main biosynthetic pathways include:

• Shikimic acid pathway ………. (phenylpropanoids).

• Mevalonic acid pathway …….. (Sterols & triterpenes).

• Amino acid pathway ………….. (alkaloids).

• Acetate pathway ……………….(fatty acids).

• Combined pathways …………..(flavonoids).

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Strategies developed to maximize the production of target compounds:

Over-expressing the key gene(s) involved in the biosynthetic pathway.

Blocking the competitive branches of biosynthesizing target compounds

increase the biomass of vagetation growth, and increase the production of target compounds.

Introduce key genes into microbes and use combinatorial biosynthesis to produce target compounds or important intermediates.




• Elicitors are substances that can trigger the hypersensitive

reaction in treated plant cells. Due to the effective up-regulation

of genes expression, and activation of secondary metabolism.

• elicitors are used widely in medicinal plant cell and tissue

culture to maximize the production of target compounds.

• Biotic elicitors: include fungal polysaccharides, proteins, cell

debris and conidium.

• Abiotic elicitors: include heavy metals ions,, UV lights, osmotic

stress and even sonication have all been reported to have

positive effects towards improvement of secondary metabolites.

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Elicitors and signaling pathways




• To improve the yield of secondary metabolites in plant

cell culture, precursor feeding is an effective approach.

• Precursors are compounds existed in upstream of

target compounds in biosynthetic pathway and most

intermediates can be used as precursors.

• The concentration of precursors determines the

reaction speed. At higher concentration, the reaction

speed is usually higher than that when precursor

concentration is lower.

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Precursor feeding




Plant name Active ingredients Culture type Reference

Taxus spp. Taxol Suspension Malik, S., et al. (2011), "

Process Biochemistry

46(1): 23-34.

Capsicum annuum Capsaicin Suspension Johnson et al., 1990,

Plant Sci. 70: 223-229.

Catharanthus roseus Indole alkaloids Suspension

Moreno et al., 1993,

Plant Cell Rep. 12: 702-

705.

Ephedra spp. L- Ephedrine Suspension O’Dowd et al., 1993,

Plant Cell Tiss. Org.

Cult. 34: 149-155.

Cassia acutifolia Anthraquinones Suspension Nazif et al., 2000,

Fitoterapia 71: 34-40.

Coffea arabica Caffeine Callus Waller et al., 1983, Plant

Cell Rep. 2: 109-112.

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Bioactive secondary metabolites from plant tissue

cultures.

Hussain, M. S., et al. (2012). "Current approaches toward production of secondary plant

metabolites." Journal of pharmacy & bioallied sciences 4(1): 10.

• Taxol, a complex diterpene anticancer alkaloid drug

found in 1971, by Wani et al. from the Pacific yew tree,

Taxus brevifolia.

• At present the drug is approved

For clinical treatment of ovarian

and breast cancer by the FDA.

• However, the supply of taxol for clinical use is limited

from either its natural source or by total synthesis.

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Production of Taxol from Taxus Sp.

Vanisree, M., et al. (2004). "Studies on the production of some important secondary

metabolites from medicinal plants by plant tissue cultures." Bot. Bull. Acad. Sin 45(1):

1-22.

Optimization of culture conditions:

• Dark conditions are suitable for the growth of cells and

taxol Production.

• A biotic elicitor from Rhyzopus stelonifera fungus

(25mg/L) used in combination with the abiotic elicitors

methyl jasmonate (10mg/L) and salicylic acid

(100mg/L)was shown to improve taxol production 16-fold

when added at day 25–30 of culture to a growth

medium.

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Malik, S., et al. (2011). "Production of the anticancer drug taxol in Taxus baccata

suspension cultures: a review." Process Biochemistry 46(1): 23-34.

• Supplementation of the medium sucrose, phenylalanine

and ammonium citrate resulted in 5.6-fold higher taxol

production (13.75mg/L) compared with the control

(2.5mg/L).

• Currently, bioreactors of up to 75,000 L are being

employed for the commercial production of paclitaxel

from cell cultures by Phyton Biotech, ESCAgenetic,

Samyang Genex, Nattermann (Germany)

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Malik, S., et al. (2011). "Production of the anticancer drug taxol in Taxus baccata

suspension cultures: a review." Process Biochemistry 46(1): 23-34.

• Capsaicin is an alkaloid obtained from Capsicum spp.

• It is used mainly as a pungent food additive in formulated

foods.

• Capsaicin is also used in pharmaceutical preparations as a

digestive stimulant and for rheumatic disorders.

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Production of Capsaicin



1-22.

• Suspension cultures of Capsicum frutescens

produce low levels of capsaicin, but immobilizing the

cells in reticulated polyurethane foam can increase

production approximately 100- fold.

• Supplying the medium with 2,4-D (2 mg/l), Kinetin

(0.5 mg/l) and Sucrose (3%) are the optimum

condition for capsaicin production.

• Further improvements can be brought about by

supplying precursors such as isocapric acid.

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1-22.

Thanks

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plant tissue culture ppt

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