Lec# 11 Plant tissue culture, GE Plants and applications Dr. Shah Rukh Abbas 11-2-2015

Agrobacterium-mediated gene transfer

How do we find “transformed” plant cells? Selectable markers in yeast and Chlamydomonas, one uses nutritional markers to complement auxotrophic phenotypes of laboratory strains (leu, trp, his, ura) in E. coli, one usually uses antibiotic resistance markers (kan, tet, chl, str, amp) in plants, one uses antibiotic or herbicide resistance markers (kan or other aminoglycosides, one of several herbicides) selectable markers used in plants are foreign genes - derived from bacteria, usually; they must be specifically tailored for expression in plant cells

Making gene transfer work Expression vectors as with selectable markers, foreign genes must usually be modified so that they can be expressed in plants most variation in expression vectors is seen in promoters constitutive or regulated promoters regulation may be “natural” - promoters derived from plant genes with desirable expression characteristics deliberately designed; constitutive or basal promoters may be specifically altered to produce a desired expression characteristic a variation on this theme, plant promoters can be modified to contain sequences recognized by bacterial or eukaryotic transcription factors tet repressor (tetracycline-inducible) lac repressor (galactoside-inducible) ACE1 activator (copper-dependent) glucocorticoid activator (glucocorticoid-inducible) These systems can be “turned on” with the application of a specific inducer (usually benign) in order for these to work, the plant must also contain the corresponding transcription factor. in order for these to work, the plant must also contain the corresponding transcription factor.

Agrobacterium-mediated gene transfer - the keys to make a segment of DNA that contains a selectable marker and a gene of interest to look like a T-DNA to get this “T-DNA” into an Agrobacterium cell so that it can be mobilized by the vir genes to produce and find transformed plant cells that can be regenerated into normal, fertile plants. -requirements a transfer cassette bounded by functioning borders ways to get this cassette into Agrobacterium disarmed Ti plasmids that retain functional vir genes

Agrobacterium-mediated gene transfer Advantages technically simple yields relatively uncomplicated insertion events (low copy number, minimal rearrangements) unlimited size of foreign DNA efficient (for most plants) adaptable to different cell types, culture procedures (protoplasts, tissue sections, “non-culture” methods) transformants are mitotically and meiotically stable Disadvantages host range is limited: not all plants may be susceptible to Agrobacterium with susceptible plants, accessible culture/regeneration systems must be adaptable to Agrobacterium-mediated gene transfer

Plant Tissue Culture Tissue culture is the term used for “the process of growing cells artificially in the laboratory” Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture) Plant tissue culture is the cultivation of plant cells, seeds, organs, tissues or protoplasts on specifically formulated nutrient enriched media in sterile conditions.

Requirements Appropriate tissue (some tissues culture better than others) A suitable growth medium containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture Growth regulators - in plants, both auxins & cytokinins. In animals, this is not as well defined and the growth substances are provided in serum from the cell types of interest Frequent subculturing to ensure adequate nutrition and to avoid the build up of waste metabolites

Basis for Plant Tissue Culture Two Hormones Affect Plant Differentiation: Auxin: Stimulates Root Development Cytokinin: Stimulates Shoot Development Generally, the ratio of these two hormones can determine plant development:  Auxin ↓Cytokinin = Root Development  Cytokinin ↓Auxin = Shoot Development Auxin = Cytokinin = Callus Development

Control of in vitro culture Cytokinin Leaf strip Adventitious Shoot Root Callus Auxin

Factors Affecting Plant Tissue Culture Growth Media Minerals, Growth factors, Carbon source, Hormones Environmental Factors Light, Temperature, Photoperiod, Sterility, Media Explant Source Usually, the younger, less differentiated explant, the better for tissue culture Different species show differences in amenability to tissue culture In many cases, different genotypes within a species will have variable responses to tissue culture; response to somatic embryogenesis has been transferred between melon cultivars through sexual hybridization Emphasize the implications for genetic involvement: Could there be undesirable genes linked to genes influencing tissue culture response?

Three Fundamental Abilities of Plants Totipotency the potential or inherent capacity of a plant cell to develop into an entire plant if suitably stimulated. It implies that all the information necessary for growth and reproduction of the organism is contained in the cell Dedifferentiation Capacity of mature cells to return to meristematic condition and development of a new growing point, follow by redifferentiation which is the ability to reorganize into new organ Competency the endogenous potential of a given cells or tissue to develop in a particular way

Culturing (micropropagating) Plant Tissue - the steps Step I Selection of the plant tissue (explant) from a healthy vigorous ‘mother plant’ - this is often the apical bud, but can be other tissue This tissue must be sterilized to remove microbial contaminants

Step II Establishment of the explant in a culture medium. The medium sustains the plant cells and encourages cell division. It can be solid or liquid Each plant species (and sometimes the variety within a species) has particular medium requirements that must be established by trial and error

Step III Multiplication- The explant gives rise to a callus (a mass of loosely arranged cells) which is manipulated by varying sugar concentrations and the auxin (low): cytokinin (high) ratios to form multiple shoots The callus may be subdivided a number of times Dividing shoots Warmth and good light are essential

Step IV Root formation - The shoots are transferred to a growth medium with relatively higher auxin: cytokinin ratios The bottles on these racks are young banana plants and are growing roots

Step V The rooted shoots are potted up (deflasked) and ‘hardened off’ by gradually decreasing the humidity This is necessary as many young tissue culture plants have no waxy cuticle to prevent water loss Tissue culture plants sold to a nursery & then potted up

Applications: Why do we Plant Tissue Culture? A single explant can be multiplied into several thousand plants in less than a year - this allows fast commercial propagation of new cultivars Taking an explant does not usually destroy the mother plant, so rare and endangered plants can be cloned safely Once established, a plant tissue culture line can give a continuous supply of young plants throughout the year In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plants

Applications: Why do we Plant Tissue Culture? Plant ‘tissue banks’ can be frozen, then regenerated through tissue culture Plant cultures in approved media are easier to export than are soil-grown plants, as they are pathogen free and take up little space (most current plant export is now done in this manner) Tissue culture allows fast selection for crop improvement - explants are chosen from superior plants, then cloned Tissue culture clones are ‘true to type’ as compared with seedlings, which show greater variability

Types of In Vitro Culture Culture of intact plants (seed and seedling culture) Embryo culture (immature embryo culture) Organ culture 1. shoot tip culture 2. root culture 3. leaf culture 4. anther culture Callus culture Cell suspension culture Protoplast culture

Tissue Culture Applications Micropropagation dihaploid production Protoplast fusion Genetic engineering