6. Cell culture and SomaclonalVariationsNew

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Plant Biotechnology Laboratory CCS HAU Regional Research Station, Karnal Dr. Ashok K. Dhawan

Plant Tissue Culture :

Plant Tissue Culture Cell culture and Somaclonal Variations

Types of In Vitro Culture Systems:

Types of In Vitro Culture Systems “ Tissue culture ” is a general term for in vitro culture of any plant part at any level of organization. This includes protoplasts, cells, callus or excised organs. Therefore tissue culture covers many types of in vitro culture systems.

Other Common Culture Systems….:

Other Common Culture Systems…. Cell Culture Protoplast Culture Organ culture Embryo culture, Meristem or shoot tip culture Anther/ovule culture Somatic embryos In Vitro Preservation Systems Transformation and Gene Technology Systems

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Ability of individual cells to grow and divide was recognized by Schwann (1839) when he proposed the Cell Theory Multiplication of single cells was done by Vasil and Hildebrant in 1965 . Plant Cell Cultures

Callus raised from an explant is introduced into liquid media (2-3 g callus/ 100 ml) and agitated to form a cell suspension.:

Callus raised from an explant is introduced into liquid media (2-3 g callus/ 100 ml) and agitated to form a cell suspension. Cell Cultures: General Procedure

Callus Development:

Callus Development Consists of unorganized dividing cells Callus cells are highly differentiated : Have a large central vacuole and nucleus to the side . Meristemetic cells are undifferentiated, small, isodiametric lack a prominent vacuole and have a large nucleus . Meristemetic cells can sometimes initiate callus masses called “ Meristemoid regions ”, which may for shoots, roots or somatic embryos.

Callus growth and Development:

Callus growth and Development Callus is a plant’s response to injury: cells around cut areas dividing to form a layer over the wound. In the process specialized cells begin to divide . Cells around the injured areas form a layer over the wound Exogenous auxin supply causes continued cell division The above also transforms specialized cells into dividing cells

Callus Cultures:

Callus Cultures Best explants: young leaves Germinated seeds in the presence of 2,4 D may produce callus without wounds Also: Pieces of cotyledons, hypocotyls, immature inflorescences, young embryos Friable (easily broken) callus disperses into liquid media more easily than hard callus. Suspension cultures grow faster than callus and are morphologically more homogenous.

Callus Cultures:

Callus Cultures Once established callus may be used for: cell cultures, protoplast isolation, somatic embryo formation, in vitro selection, organogesis, secondary product formation Callus may arise from specific regions of explant e.g. cambial ring in carrot tissue and may directly form somatic embryos from wounded regions. This can be seen only with a dissecting microscope. Apart from the explant source, genotype and hormones, orientation of the explant may be important: cut tissue touching the media. Visual selection: Embryogenic callus (usually white to yellow type from non-embryogenic (usually yellow to brown)

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Suspension cultures are Autonomous Have evenly dispersed cells Cells are removed from constraints of natural habitats Physiologically more homogenous Controllable Products of commercial value can be produced Advantages of Suspension Cultures

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Cell populations in suspension cultures follow typical sigmoid curve of -lag phase, -exponential phase, -linear phase, -deceleration phase and -stationary phase . Cell cultures follow sigmoid curve

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Growth of cultured plant cells can be measured by fresh/dry weight count of cell number heamocytometer DNA/RNA content or Packed cell volumes: Ratio of volume of cell pellet to total volume Measurement of Growth of cell cultures

Serious Problem of Suspension Cultures:

Serious Problem of Suspension Cultures Microbes grow faster : Doubling time of microbes is in minutes and plant cells in hours Contamination can be tested by: Clouding of the media Frothing of cultures Microscopy Plate on a solid media for identification of colonies

Heterogeneity of Callus:

Heterogeneity of Callus Callus originates from mixed cell types (e.g. leaves Also, variations arise due to mutant cells (somaclonal variations) and culture procedures . Hence colour, morphology, structure, metabolism and even ploidy level of callus cells varies Frequently one cell type may dominate due to faster growth Also, variations arise due to wrong base pairing during DNA replication.

Somaclonal Variations :

Somaclonal Variations

Somaclonal Variations:

Somaclonal Variations With cultured cells, genetic heterogeneity is a rule rather than exception. This is a consequence of variations in cells of original explant or are a result of culture procedures. Some are transmitted to progeny Both are included in the term “ somaclonal variations”

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“Somaclonal variations” are defined as “ genetic and phenotypic variations among clonally propagated plants derived from a single donor clone/ parent ”.

Somaclonal Variation: Occurence:

Somaclonal Variation: Occurence somaclonal variants are rare in micropropagated plants (when multiplication is by axillary branching of shoot tips/buds ) more common during shoot organogenesis & somatic embryogenesis (esp. w/a callus phase)

Why Somaclonal Variations: Plant genome is unstable:

Why Somaclonal Variations: Plant genome is unstable Plant genome is unstable and capable of generating variability Over 75% of all DNA sequences that are longer than 50 bp are repetitive DNA : These can promote incorrect pairing of chromosomes during meiosis and prone to undergoing loss or gain Most of the above is not expressed in whole plant , however

Stress of cultured Environments:

Stress of cultured Environments The hypothesis – that tissue culture is inherently stressful to cultured plant cells environmental stress is known to cause: DNA methylation – the methylation of cytosine is known to cause gene inactivation ; this may occur during the redifferentiation process Gene amplification – can result in increased gene expression

Stress of cultured Environments:

Environmental stress is known to cause : Transpositional changes Inadequate control of the cell cycle ( errors in microtubule synthesis, spindle formation ) Role of PGRs Transient modifications of phenotype (e.g., dwarfing) Stress of cultured Environments

Why Somaclonal Variations:

Why Somaclonal Variations mutations arising during the culture process cryptic changes associated with chromosomal rearrangements: -small deletions, additions, inversions - transposable genetic elements and somatic gene rearrangements.

Why Somaclonal Variations……:

Epigenetic changes arising during the culture process -Variants which exhibit some stability, but are not due to a permanent change in genetic make up of the cell - Not transmitted through sexually reproducing progeny Why Somaclonal Variations……

Epigenetic changes:

Epigenetic change in phenotype that isn't stable during sexual propagation may or may not be stable during asexual propagation habituation (most studied epigenetic change) Epigenetic changes

Habituation…:

habituation (most studied epigenetic change) Example: Loss of exogenous requirement for a growth factor ( usu. a PGR); e.g., auxin, cytokinin habituation detection – callus may lose requirement for a PGR in the process of several transfers to fresh medium Habituation…

Epigenetic change:

characteristics often occur gradually are regularly reversible (esp. in regenerated plants) are not seed-transmitted Epigenetic change

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Are an important source of variability for agriculturally important characters. Can be easily selected by subjecting cell suspension cultures to selection pressure for several generations e.g. high NaCl levels. Chemical mutagens such as EMS or nitroso-guanidine are employed to create variability prior to selection. Somaclonal variants: source of variability

In Vitro selection procedure:

In Vitro selection procedure Suitable explants cultured to obtain callus Callus made to suspension cultures Suspension cultures are subjected to selection pressure (e. g. NaCl) Rapidly growing cultures prior to selection may be subjected to mutagens Variant clones are repeatedly subcultures and subjected to selection pressure Plants raised from selected lines again tested

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Selection of salt tolerant Jatropha lines

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Available information indicates occurrence of somaclonal variations in several crops including sugarcane, potato, tobacco, tomato and rice and for a number of agronomically important characters such as disease resistance, stress and herbicide tolerance Important Successes Occurrence is common

Disease resistance:

Disease resistance Cells raised in the presence of viruses or fungal or bacterial cells or their toxins Wilt fungus Fusarium oxysporum produces fusaric acid Cultured tomato cells challenged with fusaric acid : Plants regenerated from resistant cells possessed a single dominant gene encoding disease resistance

Environmental Stresses:

Environmental Stresses NaCl ( 1 or 2 %) for selecting salt tolerance ; PEG for drought tolerance; Ions for heavy metal tolerance such as Aluminum ; Heat and cold shocks for temperature tolerance

Herbicide resistance:

Herbicide resistance Resistance of plant cells to herbicides is generally mediated through point mutations in a single gene

Successes: Somaclonal Variation:

Successes: Somaclonal Variation True Somaclonal variations have had little success except in flax, sugarcane, potato Herbicide resistant canolas, virus and disease resistant potatoes are important successes

Applications in Crop Improvement:

Applications in Crop Improvement Improvement of existing clonal (asexually propagated) cultivars sugarcane – selections for higher yield & disease resistance potatoes – yield & disease resistance improved geraniums (esp. scented varieties) woody ornamentals (e.g., Paulownia – selection for leaf variegation

Applications in Crop Improvement:

Seed cultivar improvement Fusarium resistance in celery White-flowered Torenia Tomato cultivars with higher soluble solids, (better "flesh" color) Applications in Crop Improvement

Problems of In Vitro selection:

Problems of In Vitro selection Efficient regeneration of plants from selected cells is a problem; Prolonged cultures result in loss of morphogenetic capability Stability of selected traits is a serious problem during meiosis Desirable traits already present in the species may be lost during culture process Progeny may be chimerical rather than genetically uniform

Problems: Somaclonal Variation:

Problems: Somaclonal Variation Proven to be unstable in most cases - epigenetic inheritance

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