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Tissue culture is the term used for the process of growing cells artificially in the laboratory Tissue culture involves both plant and animal cells Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture)
Haberlandt
Carrel
The Background, II
The first commercial use of plant clonal propagation on artificial media was in the germination and growth of orchid plants, in the 1920s In the 1950s and 60s there was a great deal of research, but it was only after the development of a reliable artificial medium (Murashige & Skoog, 1962) that plant tissue culture really took off commercially
1960: Cocking isolated protoplasts from cultured cells. 1962: Murashige and Skoog developed MS media for tobacco. 1966: Guha and Maheshwari obtained first haploid plants (Delhi Univ., India) 1970: Discovery of restriction endonuclease (Daniell Nathan). Plasmids were already known. 1972-73: First recombinant molecule created by Stanley Cohen, Stanford Univ. 1974: Discovery of Ti plasmid in Agrobacterium tumefaciens (by Zaenen in Ghent Univ., Belgium) 1970-80s:Ti plasmid analysis (Nester, Seattle; Van Montagu, Ghent) 1983: First transgenic plant. (Monsanto, Ghent, Washington Univ). 1985: Leaf disk transformation method (Monsanto)
seeds
embryos
Wheat inflorescence
- auxin/ + cytokinin
Callus
+ auxin
What is needed?
Tissue culture, both plant and animal has several critical 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
What is Needed, II
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
Temperature
pH
The gaseous environment
Organic supplement
Source of carbon
Gelling Agent
Organic supplement
Source of carbon
Gelling Agent
2. Organic supplement
a) Vitamins: Only thiamine (vitamin B1) is essential for most plant cultures, it is required for carbohydrate metabolism and the biosynthesis of some amino acids,
b) Myo-inositol
Although it is not essential for growth of many plant species,
3. Source of carbon
Sugars Most plant tissue cultures are not highly autotrophic due to limitation of CO2. Therefore, sugar is added to the medium as an energy source.
Organic supplement
Source of carbon
Gelling Agent
4) Gelling agents
When semi-solid or solid culture media are required, gelling agents are used.
a. Agar Agar is the most commonly used gelling agent. Composition: Agar consists of 2 components 1.Agarose is an alternating D-galactose and 3,6-anhydro-Lgalactose with side chains of 6-methyl-D-galactose residues. 2.Agaropectin is like agarose but additionally contains sulfate ester side chains and D-glucuronic acid. Agar tertiary structure is a double helix the central cavity of which can accommodate water molecules
Advantages:
Agar is an inert component, form a gel in water that melt at 100 C and solidify at nearly 45 C Concentrations commonly used in plant culture media range between 0.5% and 1%
b) Agarose It is extracted from agar leaving behind agaropectin and its sulfate groups.
c) Gelrite
d) Phytagel
Knudsons medium
dose-dependent manner
5. They may interact, either synergistically or antagonistically, to produce a particular effect.
Auxin
Cytokinin
Gibberelin
Classification of PGRs Abscisic acid Jasmonates
Ethylene Other
Salicylic acid
Brassinosteroids
Growth Regulators
Plant Hormones
Natural (made by plants) also called hormones Synthetic (man made) Also called PGRs (plant growth regulators) Purposes: start growth, stop growth, modify growth & development
Auxins
Stem elongation Produced in tips of stems (B in photo) Migrate from cell to cell in stems
Auxins - move down shaded side of the stem and cause cells to elongate
Auxins move to lowest side and cause stem tissue to elongate stem curves upwards
Apical dominance
Auxins move down the stem from the terminal bud and inhibit growth of side shoots
Pinching
Pinching = removing the terminal bud Pinching - stops flow of auxins down the stem and allows side shoots to develop Produces bushy, well-branched crops
Root development
Auxins encourage root development in cuttings Some plants produce plenty of auxins to make rooting cuttings easy Other plants need synthetic auxins such as IBA
Gibberellins
Cell elongation and cell division Stimulate development of flowers (as in gibbing camelias) Cause internodes to stretch Produced in stem and root apical meristems, seed embryos, young leaves
Internode Elongation
Gibberellins cause internodes to stretch in relation to light intensity. High light intensity = no stretch Low light intensity = long internodes. Leaves are raised to capture light
Cytokinins
Cell division (used in tissue culture) Cell differentiation (used in tissue culture for plant organ formation) Formation of callus tissue Delay aging process in plants Produced in roots Transported through xylem Still researched
Ethylene Gas
Ethylene exposure
Thickens stems Breaks down chlorophyll Weakens cell membranes Softens cell walls
Growth inhibiting hormone Responsible for seed dormancy Responsible for closing stomata during drought
Rooting compounds
Liquid or mixed with talc
Growth Retardants
Widely used in the greenhouse industry Inhibit action of gibberellins on stem elongation
Culture Types
Explants : Sterile pieces of a whole plant from which cultures
are generally initiated
Types of explant:
Generally all plant cells can be used as an explant, however young and rapidly growing tissue (or tissue at an early stage of development) are preferred.
a) Root tip:
Root cultures can be established from explants of the root tip of either primary or lateral roots. b) Shoot tip: The shoot apical meristem from either axillary or adventitious
d) Haploid tissue
Male gametophyte (Pollen in anthers) or female gametophyte (the ovule) can be used as an explant. Haploid tissue cultures can produce haploid or di-haploid plants due to doubling of chromosomes during the culture periods.
A. Callus: Definition: It is an unspecialized and unorganized, growing and dividing mass of cells, produced when explants
This necessitates the addition of other components e.g.: vitamins and, a carbon source to the culture medium, in addition to the usual mineral nutrients. Habituation: it is the lose of the requirement for auxin and/or cytokinin by the culture during long-term culture. Callus cultures may be compact or friable.
B. Cell-suspension cultures When friable callus is placed into the appropriate liquid medium and agitated, single cells and/or small clumps of cells are released into the medium and continue to grow and divide, producing a
cell-suspension culture.
The inoculum used to initiate cell suspension culture should neither be too small to affect cells numbers nor too large too allow the build up of toxic products or stressed cells to lethal levels. Cell suspension culture techniques are very important for plant biotransformation and plant genetic engineering.
The Steps, 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
Dividing shoots
Multiplication
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
The Steps, 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
The Steps, 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