introductory biotechnology

7/30/2019 Introductory Biotechnology

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S M SyiemliehDepartment of Botany

St. Anthonys College, Shillong


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What is Biotechnology ?


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1. Plant Cell and Tissue Culture (Conventional)

Underlying Concepts

Plasticity : the ability to endure extreme conditions andpredation

Totipotency : the maintenance of Genetic Potential

2. Genetic Engineering (Modern)

Categories


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Requirements

Laboratory Space :Media Room and Culture Room

Washing & Sterilization : Glassware : Special Detergents, Autoclaving Culture Media :Autoclaving, Membrane Filtration Instruments : 95 % ethanol, flaming and cooling

Plant Material : Sodium or Calcium Hypochlorite (0.3

0.6%)for 15 30 mins

NB : All operations to be performed under aseptic conditions,preferably under the hood of a laminar air flow cabinet.

Plant Cell and Tissue

Culture


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Culture Media

Constituents:

Inorganic Nutrients : C, H, O and N, P, S, Ca, K, Mg, Fe, Mn, Cu, Zn, B,Mb Organic Nutrients : Nitrogen(including Vitamins B1, B3, B5, B6 and

Amino Acids)/Carbon Sources(Including Sucrose, Glucose, Fructose, Starchetc)/Complex Nutrients(casein hydrolysate, coconut milk, corn milk, maltextract, tomato juice, yeast extract)

Growth Hormones :

1. Auxins :facilitate cell division and root differentiation;IBA, NAA, NOA, p-CPA,2,4-D and 2,4,5-T2. Cytokinins :facilitate cell division and differentiation; BAP, 2-ip, Kinetin3. Gibberellins: induce plantlet formation; 84 known plant gibberillins out of

which GA3 is the most used

Agar : 0.8-1.0%

NB : Suspension cultures must be regularly aerated either by bubbling sterile air orgentle agitation.

Plant Cell and TissueCulture


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Culture Media

Media Selection: Start with a well known medium

Evolve a new medium through a series of experiments

Growth factors (Auxins and Cytokinins) are the most variableand are to be adjusted by using five concentrations of each (0,0.5, 2.5, 5, 10 Micromol per litre of medium)

Salts can be varied at or that of MS Medium

Sucrose (2 6 %) may be used with the best combination ofgrowth regulators to find their optimum concentrations

The pH is usually set to between 5.0 and 6.0



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Suspension Culture

Cell Isolation:

Cultured Tissues Continuous agitation of calli in a liquid medium Intact plant organs

Mechanical (Grinding, Cleaning, Filtration and Centrifugation), Enzymatic (treating plant part with a Macerozyme)

Types: Batch Cultures Regular transfer of aliquots to fresh medium

Continuous Cultures Closed(Drain out used medium, Cells re-added after separation) Open (Harvest = addition of new medium)

Synchrony: Starvation Growth hormone starvation leads to cell arrest at G1 or G2

Inhibition DNA synthesis inhibitors leads to cell arrest at G1



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Applications

Mutant Selection:

Increased mutation frequency

No Chimaeras

Dominance not a problem in haploids

Direct Selection (Resistance against fungal toxins,, Antibiotics, Herbicides, etc)

Indirect Selection (NR deficient = Chlorate resistant, 5-MT resistant potato lines

selected for accumulation of Tryptophan, Phenylalanine and Tyrosine)



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Applications

Secondary Metabolites: Including Alkaloids, Glycosides,Terpenoids, Flavours, Perfumes, Agrochemicals etc.

Consistent Yield (2 10 times that of mother plant providedphysiological and Biochemical conditions are manipulated)

Consistent Quality

Predictable and Controllable production schedule

Entire process can be AUTOMATED



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Applications

Biotransformations: Low cost precursors used as a substrate toproduce High Cost Products.

Suspension culture ofDigitalis lantana can convert digitoxin into themedically important digoxin which is useful in treating heart diseases.

Daturacell cultures can convert hydroquinone into arbutin which isused as a diuretic and urinary antiseptic.

Cell cultures of Stevia rebaudiana andDigitalis purpurea can convertsteviol into steviobiocide and steviside both of which are 100 times

sweeter than cane sugar.



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Tissue Culture

Totipotency :De-differentiation forms Callus which canRe-differentiateto form the whole plant.

Explant : Pieces of differentiated tissues



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Cytodifferentiation

Auxin and Sucrose play a major role in vasculardifferentiation.

Cytokinins and Gibberellins promote differentiationinto xylem tissue.

NB :There is an inverse relationship between auxin concentration and thedegree of xylem differentiation which is dependent on sugar concentrationsince the relative amounts of xylem and phloem formation can be altered byvarying the sucrose concentration. It has been demonstrated that if auxinconcentration is constant (0.55 mg/L), then increase in sugar concentrationwill induce greater percentage of xylem formation relative to phloem.

Cell and OrganDifferentiation


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Organogenic Differentiation Shoot bud differentiation (MONOPOLAR). No general recipe can be prescribed

Cytokinin/Auxin ratio determines the shoot/root initiation in Tobacco In other plants, concentration of Auxin and not the Ratio was important

In cereals, organogenesis requires transfer from 2,4-D media to one lacking it or where2,4-D was replaced by IAA or NAA

Size of explant also matters where larger initiates shoot and smaller initiates rootformation

Somatic embryogenesis (BIPOLAR). Any part of sporophyte can give rise to an embryo. In nature it doesnt happen outside

the ovule.

Embryogenesis is influenced by plant extracts, growth regulators and calliphysiological state.

2,4-D inhibits embryogenesis in cabbage or carrot.

Embryogenesis is easy with explants grown on Auxin containing media.

Cytokinins and Gibberillins cause partial or complete inhibition

Cell and OrganDifferentiation


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MAJORBENEFITS Rapid multiplication of superior clones and maintenance of uniformity

Multiplication of disease free plants

Multiplication of sexually derived sterile hybrids

STAGES

1. Establishment of tissue in-vitro

2. Multiplication of shoots (without changing media)

3. Root formation and conditioning of propagules prior to transfer to

greenhouse(requiring alteration of media)

4. Growth in pots followed by field trials

Micropropagation


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Applications Synthetic seeds : Somatic embryos encapsulated in a

suitable matrix (eg. Sodium Alginate)o Little to No viability loss for 1 year

o Ease of handling

o Hardening not required

Virus Free Plants : Derived fromo Virus free plants

o Meristems which are generally free of infection

o Heat shocked Meristems (34-36 C)

o Callus which is usually virus free like Meristems

Micropropagation


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APPLICATIONS

Maintenance of Male Sterile parentso The F1 hybrid is fertile, while segregating F2 lines cannot be selected for sterility

until mature

o Sterile lines can therefore be micro propagated to solve this problem

Propagation of Hybrid Plants :o Male sterility is not available

o Hybrid seed production is expensive

o F2 undergoes segregation and loss of vigour

o Hybrid once obtained can be mass multiplied and can cost less

Micropropagation


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APPLICATIONS

Overcoming Crossing Barriers

o Pre-Fertilization

o Post-Fertilization

Micropropagation


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Overcoming Crossing Barriers Pre-Fertilization barrier causes

o

Differences in flowering timeo Lack of Stigma receptivity

o Lack of Pollen viability

o Failure of Pollen tube to reach the ovule due to slow growth

o Cross incompatibility due to other unknown reasons

o METHODS

1. In-vitro pollination on stigma, placenta or ovules of an excised ovary cultured on an artificialmedium depending on barrier

2. SHOTGUN WEDDING : fusion of corn egg cell and sperm to form a zygote in a test tubeusing a short pulse of electricity (Erhard Kranz and Horst Lorz, Germany)

3. Non electrical fusion methods are currently under development and testing

NB : Self incompatibility can also be overcome thus allowing selfing in hitherto self-incompatible

plants

Micropropagation


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Overcoming Crossing Barriers Post-Fertilization barrier : Embryo forms but aborts at an early stage of

development such that seeds are not formed. EMBRYO rescue must therefore

be performed.

o METHODS

1. EMBYRO Culture : Embryos are excised and cultured in appropriate media under

suitable temperature, photoperiod and humidity conditions. Most extensively used in

distant hybridizations of the family Poaceae within the tribe Triticeae.

2. OVULE Culture : Interspecific/Intergeneric Crosses involving the families Malvaceae,Fabaceae, Cruciferae, Solanaceae etc., have their Ovules excised prior to embryo abortion

and then cultured.

3. OVARY Culture : For Interspecific/Intergeneric Crosses including Brassica, Ovaries are

usually excised at the zygote or two-celled pro-embryo stage. Fruit development may be

promoted by application of IAA or Coconut milk to the medium.

Micropropagation


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OTHERUSES

1. ENDOSPERM Culture :which being triploid in its chromosome constitution is usefulfor seedless fruit production(Apple, Watermelon, Banana) production as well as triploid

production for cytogenetic studies.

2. NUCELLUS Culture : In Citrus, adventive embryos(EMBRYOIDS) develop from the

nucellar cells and given suitable culture conditions can be used to obtain plantlets.

3. GERMPLASM Storage: NBPGR, IBPGR etc maintain germplasm by storage of

tissues in culture and subsequently suspending growth by

a) Lowering the temperature

b)Adding retardants or hormones

c) Reduction in oxygen concentration

Micropropagation


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DEFINITION : Considerable variation(genetic or epigenetic) that arise from regeneration via callus,

leaf explants, or plant protoplasts which includes aneuploids, sterile plants and

morphological variants, sometimes involving economically important traits in crop plants.

CAUSES: Unknown, although variation in structure and number of chromosomes has been

suggested as one possible basis. Polyploidy, aneuploidy, translocations, inversions and

deletions have been reported in several cases. Meiotic crossing over involving symmetric and

asymmetric recombination could also be responsible.

APPLICATIONS : Recovery of Late and Early Blight resistant potato plants, Eyespot disease, Fiji

disease and Downy Mildew resistant sugarcane, etc.

APPROACHES

1. Selection is exercised in CELLS cultured for different periods and screened for the derived traits. (eg. For

resistance to specific herbicides, fungal toxins, pollutants, extremes of temperature and salinity)

2. Selection is exercised at the phenotypic level in regenerated plants.

Somaclonal Variation


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METHODS :

1. Anther or Pollen, Ovule Culture : Anthers are preferable over pollen (although embryoids areproduced from both which can be used to regenerate HAPLOID plantlets) since extraction and

culture methods for pollen differ and have been successful only in a few species. Haploids from

ovule culture were first produced from gymnosperms like Zamia, Ephedraand some Cycads. Later

on HAPLOIDS have been successfully generated for Angiosperms like barley, wheat and tobacco.

2. Chromosome elimination following interspecific hybridization (bulbosum technique) : Hordeum

vulgarex H. bulbosum(both 2n=14) yielded 95% HAPLOIDS and the remainder 5% DIPLOID

barley hybrids. Crosses between 2x H. vulgareand 4x H. bulbosumyielded TRIPLOID hybrids.

Wheat crosses with H. bulbosum(2x and 4x) also yielded viable HAPLOIDS which has led to

further exploration of such crosses in wheat breeding. Rye crosses with H. bulbosumhave also been

successful and showed the same HAPLOIDIZATION process as in the Wheat Barley crosses.

USE of HAPLOIDS : generation of HOMOZYGOUS DIPLOID LINES without SELFING

Haploids


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ISOLATION :

1. Mechanical : Rarely used nowadays due to poor yield of protoplast.. Involves plasmolysis of cells,

subsequent cutting with a fine knife.and deplasmolysis.

2. Enzymatic : Involves sterilization, peeling of epidermis, enzymatic treatment and isolation andcleaning of protoplasts. Enzymatic treatment may be:

1. DIRECT (ONE STEP) : Pectinase and Cellulase simultaneously

2. SEQUENTIAL (TWO STEP) : Pectinase followed by Cellulase

PURIFICATION :

1. Sedimentation and Washing : Centrifugation at low speed followed by resuspension in culturemedia and mannitol, then washed; the process is repeated 34 times.

2. Flotation : Concentrated Solution of mannitol, sorbitol and sucrose used as a gradient where after

centrifugation at appropriate speed, the protoplasts which are lighter than celleular debris will float

at the top and can be pipetted out.

PROTOPLAST


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METHODS :

1. Spontaneous :Yields strictly INTRASPECIFIC homokaryons or homokaryocytes each with 2-40 nuclei.

2. Induced : Used for somatic hybridization (Inter-Specific or Intra-Specific) and requires a fusogen.

Plant Specific fusogens are :

1. NaNO3 : Successfully used in oats and maize

2. Ca++ at high pH : High pH may be toxic in some cases

3. Poly Ethylene Glycol : At present, this is the best since it gives excellent results in unrelated plant taxa fusions(soybean/tobacco,soybean/maize, soybean/barley), unrelated animal taxa fusions and even in animal-plant fusions.

4. Electrical : Application of extremely short, square wave electric shock

Protoplast Fusion


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CYBRIDS : Hybridswhere NUCLEUS is derived from one parent and CYTOPLASM from

BOTH

METHODS :

1. Fusion of normal protoplast from one parent with enucleated protoplast of the other

2. Fusion of normal protoplast of one parent with protoplast containing non-viable nuclei from the

other

3. Selective elimination of one of the nuclei form the heterokaryon

4. Selective elimination of chromosomes of one parent at a later stage after fusion of the nuclei

Protoplast Fusion

introductory biotechnology

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