Molecular Plant Breeding & Tissue Culture Techniques

Application of Tissue culture in Plant Breeding BY; Zulfiqar Nasir Hussain (M.phil Students)

National University of Agricultural Sciences (NUAS), Islamabad

What is Plant Tissue Culture?  Plant

tissue culture is a form of asexual propagation of plants under laboratory conditions.

 It

is grown on a special culture medium which supports its growth and development.

 The

medium can be either semisolid, such as agar, or liquid, such as purified water.

Microorganisms and Plant Tissue Culture 

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In the practice of plant tissue culture, microorganisms are called “contaminants” because of their harmful effects on plant growth in vitro. Six potential sources of contamination in the plant tissue culture lab are:  Air  Water  Growth Media  People  Equipment  Plant Material

Why is Plant Tissue Culture Important?  It

has made significant contributions in:  The production of plant material  Plant breeding  Gene banks  The production of chemical compounds

What is Plant Breeding?  Is

a genetic improvement in a heredity material of a plant in such a way that newly formed plants are better than existing one.  A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters e.g. herbicide resistance/tolerance

Applications to Plant Breeding  Haploid

Production  Triploid Production  In Vitro Pollination and Fertilization  Somatic Hybridization & Cybridisation  Genetic Transformation  Somaclonal and Gametoclonal Variant Selection

Application to Horticulture and Forestry  

Production of Disease-Free Plants Clonal Propagation

General Applications  Secondary Metabolite Production  Germplasm Conservation

Haploid Plants from Tissue Culture   

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New Plant Varieties in a Shortened Time Frame Production of haploid plants by anther and microspore culture techniques Production of homozygous diploid lines through chromosome doubling, thus reducing the time required to produce inbred line Uncovering mutations or recessive phenotypes

Triploid Production by Plant Tissue Culture 

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Triploid plants, with three sets of chromosomes, are seed-sterile due to disturbance in gamete formation Tissue culture to morphogenic response and production of triploid plantlets have done by endosperm culture. Endosperm tissue often shows a high degree of chromosomal variations and polyploidy Triploids are usually seed sterile Seed-less ness is improve the quality of fruits

In vitro pollination and Fertilization 

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Male and female gametes are isolated and introduced to each other under conditions suitable for zygote formation In plant breeding embryo culture have been valuable tools, especially for the transfer of disease resistance genes from wild relatives into crop plants It is used for recovery of hybrid plant To protect crops from weather, pests, and diseases.

Somaclonal and Gametoclonal Variant Selection 

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Somaclonal variation and gemetoclonal variation are the important source of introducing genetic variation These variations include chromosome aberrations, DNA amplification, and the occurrence of transposable elements. Applicable to agriculture and industry specially resistance to herbicide, pathotoxin, salt or aluminium, useful in the synthesis of secondary metabolites on a commercial scale,

Somatic Hybridization and Cybridisation through Tissue Culture 

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Non-conventional genetic procedure involving fusion between isolated somatic protoplasts under in vitro conditions and subsequent development of their product (heterokaryon) to a hybrid plant is known as somatic hybridization Protoplast technology Cybridization. In cybridization, heterozygosity of extra chromosomal material can be obtained, which has direct application in plant breeding.

Genetic Transformation  Tissue

culture is an essential part of many genetic transformation protocols like; Biolistics, Agrobacterium tumefaciens, Electroporation & Microinjection  In plant breeding many different explants can be used, depending on the plant species and its favored method of regeneration

Application to Horticulture and Forestry  Production  Clonal

of Disease-Free Plant

Propagation or Micropropagation

Production of Disease-Free Plant Meristem Culture  Is a zone of cells with intense divisions; about 0.1 mm in diameter, situated in the top of buds, and extremities of roots Meristem and usually involves the removal of the meristem and subsequent culture on a nutrient medium  Pioneered Morel (1960)  Large numbers of disease-free plants may be produced from meristematic explants  stem-disc dome (SD dome) culture 

Clonal Propagation or Micro-propagation 

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A method of asexual propagation used by commercial growers to produce clones of a particular plant in large quantities Major Benefits: Rapid multiplication of superior clones and maintenance of uniformity; Multiplication of disease free plants and Multiplication of sexually derived sterile hybrids

Stages in clonal propagation:  

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Stage I involves establishment of tissue in vitro. Stage II involves multiplication of shoots (often media are not changed between stage I and stage II). Stage III concerns root formation and conditioning of propagates prior to transfer to the green house; this stage requires alteration of media for promotion of root formation. Stage IV involves growth in pots followed by field trials.

General Applications Secondary Metabolite Production 

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Produce a proliferation of shoots and leafy material, and harvesting the leafy material and shoots while in a green, actively-growing, non-senescent stage Particular interest is plant material that produces alkaloids, especially material from poppy (Papaver sp.)

Germplasm Conservation:  Genetic

resources of recalcitrantseed and vegetatively propagated species as well as of endangered species  elite genotypes which are multiplied on a large scale in production laboratories  cultures with special attributes, e.g. Metabolite-producing cell lines and genetically engineered

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