CELL CULTURE methodology Callus Cell suspension Cultures Types

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CELL CULTURE methodology Callus & Cell suspension Cultures

CELL CULTURE methodology Callus & Cell suspension Cultures

Types of in vitro cultures • • • 1. 2. 3. 4. 5. 6.

Types of in vitro cultures • • • 1. 2. 3. 4. 5. 6. Callus cultures Cell culture Protoplast culture Embryo culture Seed culture Organ culture

Callus • An unorganized mass of thin-walled parenchyma cells • Formation of wound callus

Callus • An unorganized mass of thin-walled parenchyma cells • Formation of wound callus is observed in all groups of living organisms • In Plant T. C. , callus is produced by cultivation of plant tissue on nutrient media under in vitro conditions • Presence of growth hormones in the culture media promotes callus formation and proliferation

Callus Nicotiana tabacum

Callus Nicotiana tabacum

Role of Callus • In vitro callus provides totipotent cells for plant regeneration via

Role of Callus • In vitro callus provides totipotent cells for plant regeneration via organogenesis or somatic embryogenesis • Callus is used as a target tissue for genetic transformation • Callus formation is initiated for plant regeneration of other transformed tissues • Dispersal of friable callus into single cells is used for the initiating cell suspension cultures

Initiation and maintenance of callus cultures (1) Selection of suitable parent material (2) Choice

Initiation and maintenance of callus cultures (1) Selection of suitable parent material (2) Choice of explant and method of isolation (3) Culture medium and conditions required (4) Optimization of culture conditions

 • (1) Selection of suitable parent material • Parent plant must be healthy

• (1) Selection of suitable parent material • Parent plant must be healthy and free from decay or disease • Mother plant be actively growing and not be about to enter a period of dormancy • (2) Choice of explant and method of isolation • Any part of mother plant; Plant organs or specific plant tissue or plant cells • Explant must contain living cells • Younger tissue is more callus responsive due to the presence of large no. of actively dividing cells • Explants isolated in sterile conditions • Ensure proper sterilization methods for a particular explant

(3) Culture medium and conditions required • • • Culture the sterilizd explants on

(3) Culture medium and conditions required • • • Culture the sterilizd explants on suitable autoclaved culture medium Incubate cultures at 22 -24 ± 2°C in light or dark Most callus will be initiated from the cut surfaces within 3 -8 wks. (4) Optimization of culture conditions • • • Consult literature to know previous callus initiation attempts for species under consideration For pioneering callus culture attempt, modify medium previously used for a related species Start with one of the defined media and manipulate hormone concentrations Set up a growth Latin Square of 25 culture plates with 5 each of auxin and cytokinin conc. Callus for this growth trial should be uniform and of large (20 mg) size.

Callus growth measurement • Subculture vigorous growing callus (2 -5 mm dia. ) •

Callus growth measurement • Subculture vigorous growing callus (2 -5 mm dia. ) • Slow growing callus plus explant transferred to fresh medium • Callus growth assessment via fresh and dry wts. • Plot a growth curve • Ways to improve callus growth

Latin Square arrangement for testing media combinations of auxin and cytokinins Cytokinin Conc. mg/l

Latin Square arrangement for testing media combinations of auxin and cytokinins Cytokinin Conc. mg/l Auxin Conc. mg/l 0 0. 5 02. 5 5. 0 10. 0 0 1 2 3 4 5 0. 5 6 7 8 9 10 2. 5 11 12 13 14 15 5. 0 16 17 18 19 20 10. 0 21 22 23 24 25

Cell Suspension Cultures • Cell suspension cultures are rapidly dividing suspensions of cells grown

Cell Suspension Cultures • Cell suspension cultures are rapidly dividing suspensions of cells grown in liquid medium. • Grow more rapidly than callus cultures and more amenable to culture manipulations • Comprised of cell aggregates and dispersed single cells

7 - 2. Techniques of cell culture 7 - 2 - 1. Initiation of

7 - 2. Techniques of cell culture 7 - 2 - 1. Initiation of cell suspension culture Fragments of undifferentiated callus ↓ Liquid medium ↓ ↓ ↓ aeration -------↓ Subcultureagitation -------↓ ↓ ↓ Suspension cell cultures screen small size clumps 2 - 3 g / 100 m. L

Initiation of Cell Suspensions • Initiation and establishment time depends on plant species and

Initiation of Cell Suspensions • Initiation and establishment time depends on plant species and growth medium • Dicots are easier to establish in suspensions than monocots • Initiated by agitating a fragment of callus in a vol. of liquid medium on a shaker • Three procedures used for cell dispersion: – – – Initiate from friable callus non-friable callus Callus treated with cell wall degrading enzymes

Initiation from Friable Callus • Most commonly used starting material, easily fragmented during agitation

Initiation from Friable Callus • Most commonly used starting material, easily fragmented during agitation in liquid medium, achieved by: • Callus passaged on a 7 d cycle for 2 -3 wks • Ratio of auxin/cytokinins altered, increase auxin concentration • Use 2 -3 g of friable callus per 100 ml of liquid medium • Low levels of callus tissue fail to replicate • Subculture the cells to fresh medium at a ratio of 1: 1 • Filter the actively dividing cell suspensions to remove large callus aggregates to get fine suspensions

Initiation of callus and suspension cultures

Initiation of callus and suspension cultures

 • Initiation from non-friable callus – First repeatedly subculture or transfer callus fragments

• Initiation from non-friable callus – First repeatedly subculture or transfer callus fragments to semi-solid medium – Until friable, initiate and establish cell suspensions • Initiation from callus treated with enzymes – Pectinase – Cellulase (breaks down middle lamella of cell wall and separates plant cells)

Maintenance of Cell Suspensions • Variety of culture vessels available, suitable ones allow large

Maintenance of Cell Suspensions • Variety of culture vessels available, suitable ones allow large surface area to maximize gas exchange • 20 ml culture medium + 100 ml flask • 50 -100 ml in a 250 ml flask • Flasks enclosed with sterilized/dried aluminium foil caps • Shaker speed 100 -120 rpm with optimum incubation conditions

7 - 2 - 2. Small batch culture * Culture volume : in a

7 - 2 - 2. Small batch culture * Culture volume : in a small fixed volume : < 100 m. L

Growth Characteristics of Cell Suspensions • Plant cell suspensions consist of cells with diverse

Growth Characteristics of Cell Suspensions • Plant cell suspensions consist of cells with diverse morphology and state of aggregation • Two morphological types of cells can be distinguished: – Cell aggregates made up of small cells – Large and elongated single cells • Proportion of the cell type depends on the passage of culture and nature of auxin

Monitoring the Growth of Culture • • • Cell number PCV Fresh wt. and

Monitoring the Growth of Culture • • • Cell number PCV Fresh wt. and Dry wt. Cell Viability Medium conductivity & p. H Use two methods simultaneously till stationary phase

 • Cell number – # of cells/vol. determined by heamocytometer – Dilute the

• Cell number – # of cells/vol. determined by heamocytometer – Dilute the suspension to record more than 1000 cells – Disperse large cell aggregates with 10% HCl + 10% Chromic acid (Reinert & Yeoman, 1982) • Packed Cell Volume (PCV) – Transfer aliquates (10 -15 ml) of evenly mixed cell suspension in a graduated tube – Centrifuge and note PCV as ml or % of total vol occupied by cell pellet – Accurate only for fine suspensions • Fresh and Dry Wt. – Weigh freshly harvested cells (Fresh wt) – Dry wt from cells dried at 60 °C for 48 hr. • Cell Viability – Stain the cells with viability stains (Evans Blue, FDA) and count • Conductivity and p. H – Use conductivity meter, conductivity of media falls over the passage and reflects the uptake and utilization of medium nutrients – Cultures become acidic in lag phase (p. H 3. 5 -4. 5) – Normal in stationary phase (p. H 5. 00) – p. H drop due to proton production from metabolic activity and rapid removal of media constituents like phosphate that provide buffering capacity of the medium

7 - 2 - 2. Small batch culture Plateform shaker : speed : stroke

7 - 2 - 2. Small batch culture Plateform shaker : speed : stroke range : Orbital shaker 30 -150 rpm 3/4 - 3/2 in.

m. L cells / m. L medium

m. L cells / m. L medium

Growth Curve of Suspension Cultures • Plotted for fresh, dry wts. and PCV. •

Growth Curve of Suspension Cultures • Plotted for fresh, dry wts. and PCV. • Gives the length of culture cycle • Used to decide the subculture interval of suspensions – – – Lag Phase Exponential Phase Linear Phase Decelerating Phase Stationary Phase

Uses of Cell Suspensions • Study various factors and compounds affecting growth and differentiation

Uses of Cell Suspensions • Study various factors and compounds affecting growth and differentiation • Study cell division • Rapid preparation of protoplasts • Large scale production of commercial plants via somatic embryogenesis • Commercial production of secondary metabolites

Organisation of an apical meristem (growing tip) 1 - Central zone 2 - Peripheral

Organisation of an apical meristem (growing tip) 1 - Central zone 2 - Peripheral zone 3 - Medullary (i. e. central) meristem 4 - Medullary tissue Apical meristems are composed of several layers. The number of layers varies according to plant type. I n general the outermost layer is called the tunica while the innermost layers are the corpus. In monocots, the tunica determine the physical characteristics of the leaf edge and margin. In dicots, layer two of the corpus determine the characteristics of the edge of the leaf.

 • Details of slide no. 6 • (2) Choice of explant and method

• Details of slide no. 6 • (2) Choice of explant and method of isolation • Calus can be obtained from explants of almost any part of the original plant, both from plant organs (roots, leaves and stems) or from specific tissue types (pollen, endosperm, mesophyll) or plant cells (protoplasts etc). Research aims of the project dedicate which part of plant may be used • Explant must contain living cells • Younger tissue is more callus responsive due to the presence of large no. of actively dividing cells • Many research workers use seeds, sterilize and culture in vitro. The germinated seedlings will then be used as a source of roots, hypocotyl, stem and leaves. • Glasshouse-or field-grown plants provide leaves, stems and storage tissues • In vitro-germinated seedlings are a preferred explant source as they are sterile and don’t need sterization whereas, field-grown explants must be sterilzed

 • • Details of slide 7 Culture the sterilized explants on a suitable

• • Details of slide 7 Culture the sterilized explants on a suitable autoclaved culture medium • Incubate cultures at 22 -24 ± 2°C in light or dark, depending upon the plant species. e. g. , Wheat callus needs dark and potato callus needs light for initiaiton. (3) Culture medium and conditions required • Most callus will be initiated from the cut surfaces within 3 -8 wks. Initiation of potato callus normally takes one week, wheat callus 1 -2 wks, tomato callus 23 wks and onoin callus takes 7 -8 wks.

 • When a plant is wounded callus formation occurs at the cut surfaces

• When a plant is wounded callus formation occurs at the cut surfaces and is thought to be a protective response by the plant to seal off damaged tissues. Formation of wound callus is observed in all groups of living organisms. • In plant tissue cultures callus is initiated by placing a fragment of plant tissue (an explant) on culture medium under aseptic conditions. • Callus is induced and formed from proliferating cells at the cut surface of the explant tissue. • Depending on the species callus can be initiated from a variety of tissues by employing the appropriate growth medium. It can be either plant organs (roots, leaves and stems) or from specific tissue types or cells like pollen, endosperm, mesophyll, protoplasts etc. • • The in vitro formation and proliferation of callus is enhanced by the presence of growth hormones (cyto and auxins) that promote cell division and elongation.

 • • • Callus varies widely in its general appearance and physical features.

• • • Callus varies widely in its general appearance and physical features. The variation depends on the parent tissue, age of the callus and the growth conditions Callus may be white. Green or highly colored due to the anthocyanin pigments Based on the physical appearance of callus there are 2 classes of callus, type 1 and 2 callus. 1. Type 1 callus is non-friable, Regenerates somatic embryos & organs and Frequently produces leaf-like structures 2. Whereas type 2 callus is • friable, Regenerates only somatic embryos and is Undifferentiated.