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

2. Agrobacterium-mediated gene transfer

3. 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

4. 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.

5. 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

7. 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

8. 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.

9. 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

10. 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

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

12. 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?

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

21. 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

22. 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

23. 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

24. Tissue Culture Applications
Micropropagation
dihaploid production
Protoplast fusion
Genetic engineering