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PTC - C13 (Applications of Plant Tissue Culture) Cheat Sheet (DRAFT) by

Brief summary of Chapter 13 (Applications of Plant Tissue Culture) of Plant and Tissue Culture Subject

This is a draft cheat sheet. It is a work in progress and is not finished yet.


1. The commercial production of plants which uses meristem & shoot culture to produce large numbers of identical indivi­duals.
2. To conserve rare or endangered plant species.
3. A plant breeder may use tissue culture to screen cells rather than plants for advant­ageous charac­ters.
4. Large scale growth of plants in liquid culture in biorea­ctors for the production of valuable compounds.
5. To cross distantly related species by protoplast fusion & regene­ration of the novel hybrid.

Types of In Vitro Culture (explant based)

1. Culture of intact plants (seed and seedling culture)
2. Embryo culture (immature embryo culture)
3. Organ culture (Hairy Root Culture, Shoot tip and meristem culture)
4. Callus culture
5. Cell suspension culture
6. Protoplast culture

Types of In Vitro Culture

1. Seed Culture

-growing seed asepti­cally in vitro on artificial media
->I­ncr­easing efficiency of germin­ation of seeds that are difficult to germinate in vivo
->P­rec­ocious germin­ation by applic­ation of plant growth regulators
->P­rod­uction of clean seedlings for explants or meristem culture
->In vitro selection

2. Embryo Culture

-growing embryo asepti­cally in vitro on artificial nutrient media
->R­escue embryos (embryo rescue) from wide crosses where fertil­ization occurred, but embryo develo­pment did not occur
->P­rod­uction of plants from embryos developed by non-sexual methods (haploid produc­tion)
->O­ver­coming embryo abortion due to incomp­ati­bility barriers
->O­ver­coming seed dormancy and self-s­ter­ility of seeds
->S­hor­tening of breeding cycle
The advantages of growing an embryo isolated from the rest of the seed
-To remove the immature plant from the endosperm and/or cotyle­don(s) which may in particular cases prevent or modify the develo­pment of the plant.
-As a means of propag­ating species which resist attempts to use standard methods of vegetative propag­ation.
-Rescue of weak/ aborting embryos resulting from breeding/ crossing process

3. Organ Culture

-Any plant organ can serve as an explant to initiate cultures
Culture Types
a) Shoot tip culture
b) Root culture
c) Leaf culture
d) Anther­/ovary culture

3a) Shoot Apical Meristem Culture

-Shoot tip can be cultured in vitro, producing clumps of shoots from either axillary or advent­itious buds.
-This method can be used for clonal propag­ation
-Shoot meristem cultures are potential altern­atives methods for cereal regene­ration as they are less genoty­pe-­dep­endent and more efficient
1. Production of virus free germplasm
2. Mass production of desirable genotypes
3. Facili­tation of exchange between locations (produ­ction of clean material)
4. Cryopr­ese­rvation (cold storage) or in vitro conser­vation of germplasm

3b) Root Organ Culture

-can be establ­ished in vitro from explants of the root tip of either primary or lateral roots and can be cultured on fairly simple media
-growth of roots in vitro is potent­ially unlimited, as roots are indete­rminate organs
1. Production secondary metabo­lites
2. Study the physiology and metabolism of roots, and primary root to determ­inate growth patterns

Root Culture

3d) Anther­/Ovary culture

-produ­ction of haploid plants
Ovary/­Ovule Culture
1. Production of haploid plants
2. A common explant for the initiation of somatic embryo­genic cultures
3. Overcoming abortion of embryos of wide hybrids at very early stages of develo­pment due to incomp­ati­bility barriers
4. In vitro fertil­ization for the production of distant hybrids avoiding style and stigmatic incomp­ati­bility that inhibits pollen germin­ation and pollen tube growth
Anther and Microspore Culture
1. Production of haploid plants
2. Production of homozygous diploid lines through chromosome doubling, thus reducing the time required to produce inbred lines
3. Uncovering mutations or recessive phenotypes

4. Callus Culture

-un-or­ganized mass of cells
-tissue that develops in response to injury caused by physical or chemical means
-Most cells of which are differ­ent­iated although may be and are often highly unorga­nized within the tissue
Culturing Callus
-Produ­ction of plantlets through somatic embryo­genesis or organo­genesis
-Secondary metabo­lites production
-Any plant tissue can be used as an explant
-often performed in the dark as light can encourage differ­ent­iation of the callus
-During long-term culture, the culture may lose the requir­ement for auxin and/or cytokinin - ‘habit­uation’ - common in callus cultures
-Manip­ulation of the auxin to cytokinin ratio in the medium can lead to the develo­pment of shoots, roots or somatic embryos
-Callus cultures can also be used to initiate cell suspen­sions
Develo­pment of Callus
-The prolif­eration can be maintained more or less indefi­nitely, if the callus is subcul­tured on to fresh medium period­ically
-Callus is usually composed of unspec­ialized parenchyma cells
-During callus formation there is some degree of dediff­ere­nti­ation
-i.e. the changes that occur during develo­pment and specia­liz­ation are, to some extent, reversed, both in morphology and metabolism
-One major conseq­uence of dediff­ere­nti­ation: most plant cultures lose the ability to photos­ynt­hesize
Two categories of callus:
a) Compact callus: the cells are densely aggregated
b) Friable callus: the cells are only loosely associated with each other and the callus becomes soft and breaks apart easily
The friability of callus can be improved by:
a) manipu­lating the medium components
b) repeated subcul­turing
c) culturing it on ‘semi-­solid’ medium (medium with a low concen­tration of gelling agent)

5. Cell Suspension Culture

-When callus pieces are agitated in a liquid medium, they tend to break up.
-Suspe­nsions are much easier to bulk up than callus since there is no manual transfer or solid support.
-Friable callus provides the inoculum to form cell-s­usp­ension cultures
Growing of cell suspension from friable callus
->l­iquid medium
->s­ingle cells and/or small clumps of cells are released
->c­ontinue to grow and divide
->c­ell­-su­spe­nsion culture
Criteria in growing cell suspension from friable callus:
-A relatively large inoculum should be used when initiating cell suspen­sions so that the released cell numbers build up quickly.
-The inoculum should not be too large though, as toxic products released from damaged or stressed cells can build up to lethal levels.
-Cell suspen­sions can be maintained relatively simply as batch cultures in conical flasks.
-The degree of dilution during subculture should be determined empiri­cally for each culture.
-Too great a degree of dilution will result in a greatly extended lag period or, in extreme cases, death of the transf­erred cells.
-After subcul­ture, cells divide and culture biomass increases in a charac­ter­istic fashion, until nutrients in the medium are exhausted and/or toxic by-pro­ducts build up to inhibitory levels ->‘­sta­tionary phase’
-If cells are left in the stationary phase for too long, they will die and the culture will be lost
-cells should be transf­erred as they enter the stationary phase
-important to determine batch growth­-cycle parameters for each cell-s­usp­ension culture
1. it can ultimately provide a contin­uous, reliable source of natural products.
2. synthesis of bioactive secondary metabo­lites
3. running in controlled enviro­nment
4. indepe­ndently from climate and soil conditions
Importance and Applic­ation of cell suspension culture as an experi­mental technique
-Contr­ibute inform­ation about cell physio­logy, bioche­mistry, metabolic events at the level of individual cells and small cell aggregates
-Develop unders­tanding of an organ formation or embryoid formation start­ing from single cell or small cell aggregates
-Suspe­nsion culture derived from medici­nally important plants can be studied for the production of secondary metabo­lites
-Mutag­enesis studies may be facili­tated by the use of cell suspension cultures to produce mutant cell clones from which mutant plants can be raised

Introd­uction into Suspension

6. Protoplast Culture

-The living material of a plant or bacterial cell, including the protoplasm and plasma membrane after the cell wall has been removed.
Somatic Hybrid­ization
-devel­opment of hybrid plants through the fusion of somatic protop­lasts of two different plant specie­s/v­ari­eties
1. Isolation of protoplast
2. Fusion of the protop­lasts of desired specie­s/v­ari­eties
3. Identi­fic­ation and Selection of somatic hybrid cells
4. Culture of the hybrid cells
5. Regene­ration of hybrid plants
How to make a protop­last?
-Two general approaches to removing the cell wall (without damaging the protop­last):
a) Mechanical isolation: although possible, often results in low yields, poor quality and poor perfor­mance in culture due to substances released from damaged cells
b) Enzymatic isolation: usually carried out in a simple salt solution (with a high osmoticum) + cell wall degrading enzymes.
-It is usual to use both cellulase and pectinase enzymes (must be of high quality and purity)
-Proto­plasts are fragile and easily damaged -> must be cultured carefully.
-Liquid medium is not agitated and a high osmotic potential is mainta­ined, at least in the initial stages.
-The liquid medium must be shallow enough to allow aeration in the absence of agitation
-Proto­plasts can be plated out on to solid medium and callus produced
-Whole plants can be regene­rated by organo­genesis or somatic embryo­genesis from this callus
-Proto­plasts are ideal targets for transf­orm­ation by a variety of means
Uses for Protoplast Fusion:
1. Combine two complete genomes
->a­nother way to create allopo­lyp­loids
2. In vitro fertil­ization
3. Partial genome transfer
->E­xchange single or few traits between species
->May or may not require ionizing radiation
4. Genetic engine­ering
->M­icr­o-i­nje­ction, electr­opo­ration, Agroba­cterium
5. Transfer of organelles
->U­nique to protoplast fusion
->The transfer of mitoch­ondria and/or chloro­plasts between species


Protoplast Fusion


Plant Genetic Engine­ering

Genetic Transf­orm­ation
-Intro­duction of foreign DNA to generate novel genetic combin­ations.
-Transfer of desirable genes for disease and pest resistance from related or unrelated plant species into high yielding suscep­tible cultivars.
-Study of structure and function of genes.
Purposes of Introd­ucing Novel Traits
1. Biodiv­ersity screening: To look for new traits in other or wild population
2. Management of germplasm: Propag­ation of elite germplasm through microp­rop­agation
3. Inters­pecific or interg­eneric crosses: Introd­uction of traits from other genus or species
4. Overcome crossing barrier: Use protoplast fusion or somatic hybrid­ization to produce hybrids
5. Genetic engine­ering: Introduce genes from the same species, distantly related species or unrelated species (e.g. bacteria into plants)
6. Incorp­oration into plant breeding: Use selected inbreeds for gene transf­orm­ation
Creation of Novel Traits
Somaclonal variation
-Variation arise in culture (espec­ially those undergone long period of culturing) due to genetic or epigenetic mutation
-Treatment of cultures with mutagens, such as ethyl methane sulfonate (EMS), UV and radioa­ctive radiations
-Dosage of mutagens can be vital, sub-vital or sub-lethal
-Mild mutation - opt for vital
-Heavy mutation – opt for subvital to sublethal
Purposes of Creating Novel Traits
Propagate high yielders
-Micro­pro­pag­ation of high yielders could increase yield
-This could be a strategy until full inbreeds are produced
Disease suscep­tib­ility
-Reliance on one clone is dangerous to food security, since disease suscep­tib­ility could decimate the entire crop
-At least a few clones of the high yielders must be grown in a field and best if they are separated by rows

Plant Genetic Engine­ering Process

Somaclonal Variation

Germplasm Conser­vation

-Germplasm is living tissue from which new plants can be grown
(googl­e)-­Ger­mplasm is the term used to describe the seeds, plants, or plant parts useful in crop breeding, research, and conser­vation efforts.
An extension of microp­rop­agation techniques through two methods:
-Slow growth techniques e.g.: ↓ Temp., ↓ Light, media supple­ments ( growth retard­ants).
-Mediu­m-term storage (1 to 4 years)
-Ultra low temper­atures in liquid nitrogen at -196°C.
-Stops cell division & metabolic processes
-Very long-term (indef­inite)

Pros and Cons of Plant Tissue Culture

1. In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plants
1. It is a labor intensive & expensive process.
2. Plant “tissue banks” can be frozen, the regene­rated through tissue culture
2. Not all plants can be succes­sfully tissue cultured - it is usually because the medium of growth is not known.
3. Plant culture in approved media are easier to export than soil-grown plants, as they are pathogen free and take up little space (most current plant export is now done in this manner
4. Tissue culture allows fast selections for crop improv­ement – explants are chosen from superior plants then cloned
5. High degree of uniformity (true type plants) when compared to conven­tio­nally produced plants