7. Protopl and OtherCul Systm New

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

Plant Tissue Culture :

Plant Tissue Culture Protoplast and Other Culture Systems

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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I. Protoplast Culture

Somatic Cell Hybridization:

Somatic Cell Hybridization Cells have recognition mechanisms and distinguish self from non-self : The Incompatibility. Hence Unrelated plants (interspecific or intergeneric) when crossed may not produce viable seeds.

Why Incompatibility?:

Why Incompatibility? Pollen may not germinate Fertilization may not occur Embryo, endosperm or seed may be non-viable

Man’s Quest for New Solutions:

Man’s Quest for New Solutions Somatic Cell hybridization or Parasexual hybridization to produce a somatic hybrid or cytoplasmic hybrid involves Protoplast fusion technology. Protoplast is a Plant Cell without cell wall i.e. membrane + cytolplasm Cocking (1960) used cellulose-degrading enzymes from fungi to produce protoplasts.

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Tissue for protoplast isolation may come from callus, suspension cultures or plant tissues. When plant tissues are to be used, young leaves are the best source Protoplast Isolation

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Cell Walls have Pectin, cellulose, hemicellulose Pectinases degrade pectins to macerate tissue to single cells Cellulases digest cellulose and Hemicellulases break hemicellulose. Incubation with 3 enzymes releases protoplasts Protoplasting enzymes usually isolated from Fungi such as Aspergillus Protoplasting Enzymes

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After 10-20 h of incubation with 3 enzymes on a shaker protoplasts are released. Suspensions are filtered through 40-50 micron mesh The filtrate is centrifuged and pellet re-suspended in 0.7 M Mannitol Protoplast Isolation

The Plant Cell Walls:

The Plant Cell Walls Protoplasts are dispersed in a media that ensures their osmotic stability and viability. Since protoplasts are normally “ pumped up ” against cell walls, media has to have slightly higher molarity than cytosol (0.7 M Mannitol) Cultured protoplasts synthesize cell walls and undergo division to form callus

Protoplast fusion:

Protoplast fusion Protoplasts are isolated from two species that are to be to crossed The protoplast membranes are made to fuse Regenerate the hybrid fusion product Contain genome from both organisms

Fusion of Protoplasts:

Fusion of Protoplasts Membranes of the two protoplasts have to be reversibly destabilized to cause fusion Chemical Fusion : 10 to 50 % PEG causes extensive, non-specific agglutination. PEG has high affinity for water ; immobilizes water molecules in the vicinity of membranes. Fusion is caused due to decrease in hydrophobic stabilization of lipid bi layer.

Fusion…..:

Fusion….. Some protoplasts are intrinsically very fusogenic and can be induced to fuse by high pH (10.5) and high Ca ++ ion conc . PEG is used in combination with high pH (10.5) and high Ca ++ ion conc. However, PEG causes multiple fusions rather than binary fusions and is somewhat toxic and reduces regeneration

Electro Fusion:

Electro Fusion Electro fusion is holding protoplasts under electric fields to undergo fusion. Two steps: Cell agglutination : Protoplast suspension placed in a chamber with two metal electrodes Fusion is induced by a single high voltage DC pulse Adjacent membranes fuse into single continuous layer

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Heterokaryon : Only membranes fuse Hybrid Cells : Nuclear fusion also occurs Cybrids or cytoplasmic hybrids : Cytoplasm of two parents, nucleus of one parent ( enucleated protoplast) Fusion products

Applications of protoplast Technology:

Applications of protoplast Technology Production of somatic hybrids in sexually incompatible species Transfer of cytoplasmic genetic elements to produce cytoplasmic hybrids or cybrids Protoplasts may be transformed by direct DNA uptake by electroporation or PEG mediation Production of hybrid lines in species such as potato and sugarcane that normally propagate vegetatively.

II. Organ Cultures:

II. Organ Cultures Organ Culture: Cultured plant material that maintains its morphological identity as an organ with same basic anatomy and physiology as of parent plant e.g. culture of excised roots.

III. Embryo Culture:

III. Embryo Culture Washed seeds are surface sterilized and embryos removed aseptically and cultured on the media. Some embryos show erratic or no growth: - Poor food reserves as in orchid seeds -Presence of inhibitors - lack of some essential enzymes as in coconut Culture of immature orchid embryos is important in obtaining rare hybrids.

Embryo Rescue:

Embryo Rescue Culture of excised aborting embryos is called embryo rescue Some embryos show erratic or no growth and tend to abort Embryo is rescued from the plant and cultured in vitro into a viable plant Embryos beyond the globular stage of development are preferred as young zygotic embryos are difficult to culture

Embryo Rescue for Hybrid plants:

Embryo Rescue for Hybrid plants Place pollen from one species on the emasculated flower of the second species After a few days dissect Embryo out the developing seed and culture Regenerate the hybrid Triticale - developed by crossing wheat and rye

IV. Meristem, shoot or stem-tip culture:

IV. Meristem, shoot or stem-tip culture Excised shoot tips for shootlets or plantlets Proliferating areas at the base of shoot tip explant forms an “ Open-ended” system for shootlet formation Newly formed shootlets are subcultured to produce additional auxiliary branches.

Meristem culture for Virus Free Plants:

Meristem culture for Virus Free Plants There is no chemical treatment for plants that are internally infected with Viruses Viruses are generally not spread through seeds , but are a serious issue in vegetatively propagated plants : potato, garlic, banana, sugarcane strawberries etc. Heat inactivation of viruses by thermotherapy : heating the tissue in water bath or growth chambers or meristem culture or both together are employed to get virus free plants.

Meristem Culture:

Meristem Culture Apical Shoot meristem and first few primordial leaves are generally not connected to vascular tissue and hence not contaminated by Viruses Explant be carefully excised: sap from mature leaves or stem tissue should not contaminate it. True “ Shoot meristem ” is isolated apical dome with no primordial leaves. However, “ Shoot tip ” i.e. Apical dome + 2 to 4 subjacent primordial leaves is taken Shoot tip in rapid growth should be preferred. Being a microscopic explant this requires expertise and patience.

V. Anther/Ovule culture:

V. Anther/Ovule culture Anthers and Ovules are source of haploid plants Androgenesis : production of haploid plants from male gametophyte: Anthers containing immature pollen grains are cultured. Gynogenesis: production of haploid plants from female gametophyte

Haploid Culture:

Haploid Culture Culture of the plant gametes - pollen and ovule ( egg cells ) Cells are haploid Regenerated plants are haploid Regenerated plants are sterile Can’t undergo meiosis

Haploid Culture:

Haploid Culture Chromosomes need to be doubled Use mitotic inhibitors - colchicine, fluoralin Plantlets immersed in 0.5% colchicine for 24-48 h Recovered plants are diploid Recovered plants are 100% homozygous “Fixes” traits, decreased breeding time Most canola varieties are doubled haploid

Haploid Plants are Important:

Haploid Plants are Important Haploid plants possess single set of chromosomes and even recessive mutations are expressed phenotypically Doubling of chromosomes spontaneously or with colchicine results in homozygous plants Homozygous diploids important to Breeders in hybrid programmes and usually takes 3 to 5 generations to develop these.

VI. Somatic Embryos:

VI. Somatic Embryos Steward (1958) first induced “ embryoids ” in suspension cultures of carrot. Somatic embryos may be formed direct without formation of callus or induced from callus. Induction of embryogenic cells requires high auxins , but their development requires reduced levels or no auxins.

VII. In Vitro Preservation:

VII. In Vitro Preservation Germplasm storage in liquid nitrogen at -196 0 C Sterile shoot apices or callus wrapped in sterile squares of aluminium foil and plunged in liquid N. Minimal growth storage also possibly by: - sub-optimal basal media Media containing retardants (ABA, MH) Unfavorable physical conditions (0-10 oC temp)

XIII. Genetic Transformation and Gene Technologies:

XIII. Genetic Transformation and Gene Technologies Raising of callus and regeneration of transformed cells are basic requirements of gene transfer technologies

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