Cloning and Growth-regulators.

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Copyrights © Reserved Markus Axelsson, 2005.

Cloning

Cloning is defined as processes resulting in a clone; natural and artificial. A clone is a population of organisms which asexually has arised from the same organism as origin (meaning that the 'new' organisms have exactly the same genotype)

When cloning plants, one takes advantage of the fact that all plant cells are totipotent cells, meaning that the cells are able to dedifferentiate to non-specific cells and then divide further as such totipotent cells. The cells form a callus. Hormones are used to start and keep up these processes in vitro.

At higher concentrations of an auxin there will probably be callus proliferation (on-going divisions of non-specific cells). Sometimes, liquid media are used. The cultures must then be shaken during the processes to allow the cells to exchange gas sufficiently and to prevent organogenesis (specialisation to tissue) of the cells. Cells at 'nodes' (meristmatic cells) can start growing in cultures without first dedifferentiate. That's the reason to why stems of e.g. Phalaenopsis often are used to produce clones.

Explant

The explants best suited for micropropagation naturally contain meristmatic tissue cells. Leaves are also used (the younger, the better), as well as root-tips.

The surface sterilization of the explants is as important as the sterilization of seed for sowing on nutrient media. The explants may be able to handle the treatment a bit better than the seeds. Sterilant concentrations and times for sterilization are still the same as recommended, regardless of the use of explant or seed.

Replate of structures that have started growing in vitro can be done after a few months after they have started growing. The need for replate vary greatly and is of course specific for the project and the expected end product of the culturing. There is a risk that phenols are exudated by the explants. It's visible as a colouring of the medium (increased turbidity locally). The phenols may harm the cells. (Ascorbic acid has been used with success, in preventing theses exudations.)

Hormones

Hormones are chemical messengers in organisms. In plants, they regulate cell division, root growth, shoot production, and flowering (only to mention a few of the effects). These are only a couple of all the available substances. It's generally difficult finding the relations between hormones to get the regulating effect wanted. In many cases, there simply has to be studies made to ensure what concentrations to use (for technical and scientific use).

[Approx. recommended concentrations are given after the substances'[]', Source: sigma-aldrich.]

† Some substances are narcotic precursors (metabolites of narcotics). (Some restrictions prevail the use of such substances.)

Auxins:

Auxins extend cells and are important root growth regulators in cultures. Starts cell division (mitosis) in connection to cytokinins. Auxins stimulate cells to undergo embryogenesis; at higher concentrations there is a callus proliferation and at lower concentrations, the rooting effect dominates. These substances counteract axillary growth and bud formation. With gibberellines, auxin promote fruit set.

 

2,4-D 2,4-dichlorophenoxyethanoic acid [0,01-6,0mg/L]

A herbicide used mostly fight growth of certain dicotyledons (not efficient against monocotyledons). In cultures it's added to keep up a callus cell proliferation. It also suppress the differentiation (organogenesis) heavily. (Similar to 2,4,5-T, or 'Orange', which was used in Vietnam during the war.)

 

NAA a-naphtalene acetic acid [1,0mg/L]

Stimulates rooting at lower concentrations [0,5-1,0mg/L] and callus proliferation at higher concentrations; exogenic (analogous to IAA).

 

IAA indole-3-ethanoic acid up to [3,0mg/L]

Controls the organogenesis of shoot cells and cell division in roots naturally in plants; endogenic. Can start callus proliferation in cultures (at higher concentrations).

 

IBA indole-3-buturic acid [0,1-10mg/L]

Used to induce rooting of shoots (e.g. of root-free shoots derived from a culture).

 

PAA phenylethanoic acid [0,1-50mg/L] †

Extends cells and may induce a production of shoots together with cytokinins.

 

Cytokinins:

Cytokinins have the effect of increasing the mitosis (cell division) of shoot and buds. Some cytokinins increase the size of the plant cells and e.g. makes leaves larger The cell differentiation can be controlled (e.g. for production of a large amount of calli-cells). Growth of roots is inhibited. Cytokinins are adenine derivatives and kinetin can e.g. be synthesised from heating nucleic acid. Coconut water (the liquid interior of a coconut) contains cytokinins to stimulate the growth of the coconut palm.

 

2iP N⁶-(3-methylbut-2-enyl)adenine [1,0-3,0mg/L]

An efficient hormone, inducing shoot- and leaf growth, suppressing root formation.

 

Kinetin 6-furfurylaminopurine [0,1-10mg/L]

Exogenic but almost identical to natural substances. Often used in cultures. Stimulates shoot- and leaf growth at lower concentrations (callus proliferation at higher concentrations).

 

Benzyladenine N⁶-benzyladenine [0,1-10mg/L]

Synthetic cytokinin just like kinetin.

 

BA 6-benzylaminopurine [0,1-5,0mg/L]

Stimulates a production of adventious shoot and callus; see description of kinetin.

 

Zeatin [0,1-5,0mg/L]

Naturally occuring cytokinin (first isolated from corn).

 

Adenine sulfate 2C₅H₅N₅· H₂SO₄ [50-250mg/L]

 

Thidiazurone 1-fenyl-3-(1,2,3-tiadiazole-5-yl)urea [0,001-0,05mg/L]

Gibberellins and other substances:

Cells are extended (there's especially an extension of internodes) and cells get larger from gibberellins. The flower production is probably stimulated and the metabolism of many seeds are regulated by gibberellins (e.g. mobilization of energy storing substances in seeds and a stimulation of amylase production to digest starches); meaning that these substances may 'wake' certain seed up from resting stages. Seed-less grapes are grown while treated with gibberellines.

 

GA (gibberellinic acid) [0,01-5,0mg/L]

A treatment of seeds may stimulate germination. The growth of shoot is also stimulated, while root formation is inhibited.

 

CCC (2-chloroethyl)trimethylammoniumchloride [<500mg/L]

Growth inhibiting substance. May slow down the germination of seeds.

 

Ethylene CH₂CH₂

Ethylene is produced by ripening and even decaying plants. There is a reason to why one shouldn't place certain plants close to a fruit bowl.

 

Silvertiosulfate (STS) e.g. used to inhibit ethylene production in vitro.

 

ABA Abscisic acid [0,1-10mg/L]

A substance that isn't either an auxin, a cytokinin, nor a gibberellin. It's naturally produced by a plant feeling stress (inhibits growth and induces processes that save energy). (the stomata is closed - the plant respires less and lose less water). This is a 'negative factor'. ABA stimulates the growth of pseudobulbs and the maturation of embryos.

 

Oligosaccarides C₆H₁₂O₆

Various oligosaccarides may have effects on the growth of plants. (e.g. growth rate, flowering etc.)

 

Polyamines

Some polyamines have effects on the growth of plant cells and their development. Spermidine and putrescine are common. Spermidine promotes embryogenesis of somatic cells and putrescine increase the growth of adventious roots.