S M Syiemlieh

Department of Botany St. Anthonys College, Shillong

What is Biotechnology ?

Categories
1. Plant Cell and Tissue Culture (Conventional)
Underlying Concepts
Plasticity : the ability to endure extreme conditions and predation Totipotency : the maintenance of Genetic Potential

2. Genetic Engineering (Modern)

Plant Cell and Tissue Culture

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

Culture Media
Constituents:

1.

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, Starch etc)/Complex Nutrients(casein hydrolysate, coconut milk, corn milk, malt extract, tomato juice, yeast extract) Growth Hormones :
2. 3.
Auxins : facilitate cell division and root differentiation; IBA, NAA, NOA, pCPA, 2,4-D and 2,4,5-T Cytokinins : facilitate cell division and differentiation; BAP, 2-ip, Kinetin 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 or gentle agitation.

Plant Cell and Tissue Culture

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 variable and 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 of growth regulators to find their optimum concentrations The pH is usually set to between 5.0 and 6.0

Plant Cell and Tissue Culture

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

Plant Cell and Tissue Culture

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)

Plant Cell and Tissue Culture

Applications

Secondary Metabolites: Including Alkaloids, Glycosides,

Terpenoids, Flavours, Perfumes, Agrochemicals etc.
Consistent Yield (2 10 times that of mother plant provided physiological and Biochemical conditions are manipulated)

Consistent Quality
Predictable and Controllable production schedule Entire process can be AUTOMATED

Plant Cell and Tissue Culture

Applications

Biotransformations: Low cost precursors used as a substrate to

produce High Cost Products.
Suspension culture of Digitalis lantana can convert digitoxin into the medically important digoxin which is useful in treating heart diseases.

Datura cell cultures can convert hydroquinone into arbutin which is used as a diuretic and urinary antiseptic.
Cell cultures of Stevia rebaudiana and Digitalis purpurea can convert steviol into steviobiocide and steviside both of which are 100 times sweeter than cane sugar.

Plant Cell and Tissue Culture

Tissue Culture
Totipotency : De-differentiation forms Callus which can Re-differentiate to form the whole plant.
Explant : Pieces of differentiated tissues

Cell and Organ Differentiation

Cytodifferentiation

Auxin and Sucrose play a major role in vascular differentiation. Cytokinins and Gibberellins promote differentiation into xylem tissue.

NB : There is an inverse relationship between auxin concentration and the

degree of xylem differentiation which is dependent on sugar concentration since the relative amounts of xylem and phloem formation can be altered by varying the sucrose concentration. It has been demonstrated that if auxin concentration is constant (0.55 mg/L), then increase in sugar concentration will induce greater percentage of xylem formation relative to phloem.

Organogenic Differentiation

Cell and Organ 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 where 2,4-D was replaced by IAA or NAA Size of explant also matters where larger initiates shoot and smaller initiates root formation

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 calli physiological 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

Micropropagation

MAJOR BENEFITS
Rapid multiplication of superior clones and maintenance of uniformity Multiplication of disease free plants Multiplication of sexually derived sterile hybrids

STAGES
1. 2. 3. 4. Establishment of tissue in-vitro Multiplication of shoots (without changing media) Root formation and conditioning of propagules prior to transfer to greenhouse(requiring alteration of media) Growth in pots followed by field trials

Micropropagation
Applications
o o o

Synthetic seeds : Somatic embryos encapsulated in a suitable matrix (eg. Sodium Alginate)
Little to No viability loss for 1 year Ease of handling Hardening not required

Virus Free Plants : Derived from

o o o o Virus free plants Meristems which are generally free of infection Heat shocked Meristems (34-36 C) Callus which is usually virus free like Meristems

Micropropagation
APPLICATIONS

Maintenance of Male Sterile parents

o o The F1 hybrid is fertile, while segregating F2 lines cannot be selected for sterility until mature Sterile lines can therefore be micro propagated to solve this problem

Propagation of Hybrid Plants :

o o o o Male sterility is not available Hybrid seed production is expensive F2 undergoes segregation and loss of vigour Hybrid once obtained can be mass multiplied and can cost less

Micropropagation

APPLICATIONS
Overcoming Crossing Barriers
o o Pre-Fertilization Post-Fertilization

Micropropagation

o o o o o o 1. 2. 3.

Overcoming Crossing Barriers

Pre-Fertilization barrier causes
Differences in flowering time Lack of Stigma receptivity Lack of Pollen viability Failure of Pollen tube to reach the ovule due to slow growth Cross incompatibility due to other unknown reasons METHODS In-vitro pollination on stigma, placenta or ovules of an excised ovary cultured on an artificial medium depending on barrier SHOTGUN WEDDING : fusion of corn egg cell and sperm to form a zygote in a test tube using a short pulse of electricity (Erhard Kranz and Horst Lorz, Germany) 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

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

o
1.

2.

3.

Micropropagation
OTHER USES
1.

ENDOSPERM Culture : which being triploid in its chromosome constitution is useful for seedless fruit production(Apple, Watermelon, Banana) production as well as triploid production for cytogenetic studies. NUCELLUS Culture : In Citrus, adventive embryos(EMBRYOIDS) develop from the nucellar cells and given suitable culture conditions can be used to obtain plantlets.

2.

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

Somaclonal Variation

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.

Haploids

METHODS :
1. Anther or Pollen, Ovule Culture : Anthers are preferable over pollen (although embryoids are produced 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, Ephedra and some Cycads. Later on HAPLOIDS have been successfully generated for Angiosperms like barley, wheat and tobacco. Chromosome elimination following interspecific hybridization (bulbosum technique) : Hordeum vulgare x H. bulbosum(both 2n=14) yielded 95% HAPLOIDS and the remainder 5% DIPLOID barley hybrids. Crosses between 2x H. vulgare and 4x H. bulbosum yielded 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. bulbosum have also been successful and showed the same HAPLOIDIZATION process as in the Wheat Barley crosses.

2.

USE of HAPLOIDS : generation of HOMOZYGOUS DIPLOID LINES without SELFING

PROTOPLAST

ISOLATION :
1. 2. Mechanical : Rarely used nowadays due to poor yield of protoplast.. Involves plasmolysis of cells, subsequent cutting with a fine knife.and deplasmolysis. Enzymatic : Involves sterilization, peeling of epidermis, enzymatic treatment and isolation and cleaning of protoplasts. Enzymatic treatment may be:
1. DIRECT (ONE STEP) : Pectinase and Cellulase simultaneously
2. SEQUENTIAL (TWO STEP) : Pectinase followed by Cellulase

PURIFICATION :
1. 2. Sedimentation and Washing : Centrifugation at low speed followed by resuspension in culture media and mannitol, then washed; the process is repeated 3 4 times. 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 Fusion

METHODS :
1. Spontaneous : Yields strictly INTRASPECIFIC homokaryons or homokaryocytes each with 240 nuclei. 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

2.

Protoplast Fusion

CYBRIDS : Hybrids where NUCLEUS is derived from one parent and CYTOPLASM from BOTH METHODS :
1. 2. 3. 4. Fusion of normal protoplast from one parent with enucleated protoplast of the other Fusion of normal protoplast of one parent with protoplast containing non-viable nuclei from the other Selective elimination of one of the nuclei form the heterokaryon Selective elimination of chromosomes of one parent at a later stage after fusion of the nuclei