Cytokinins are degradation products of DNA. They are made up of adenine nucleus and a furfural ring. This basic structure is known as 6- furfuryl aminopurine. Cytokinins stimulate cell division and enlargement of cells. The deficiency of cytokinins results in stunted growth in plants.
Cytokinins are classified into the following categories:
Zeatin: It is a naturally occurring cytokinin in plants. Zeatin exists in two forms: One is cis- zeatin, and the other is trans-zeatin. Zeatins are produced from Agrobacterium tumefaciens, Amanita rubescens, Rhizopogon, Ochraceorubens, R. roseplus, Saccharomyces, Ustilago esculanta, etc.
Kinetin: chemically kinetin is a 6- furfuryladenine. It is present in Amanita rubescens, Nectrias galligena, Streptomyces albus, etc.
N-6 2- Isopentenyl Adenine: It is present in Agrobacterium tumefaciens, Azotobacter chroococcum, Taphrina cerasi, etc
t- RNA Ribosyl Zeatin: It is presnt in Erwinia amylovora, Rhizobium leguminisarum, etc.
Methyl Thio- ribosyl Zeatin: It is present in R. leguminosarum, A. tumefaciens, etc.
Azotobacter vinelandii, A. chroococcum, Taphrina cerasi, Rhizobium leguminosarum, Saccharomyces cerevisiae, E.coli, etc. are used in the production of cytokinin and cytokinin- like compounds.
The metabolism of cytokinin was well studied in E. coli cells. The purine nitrogen base adenine reacts with ribose-1-monophosphate to form cytokinin-9-riboside. This reaction is catalysed by the enzyme, purine nucleoside phosphorilase.
In some organisms the isopentyl group directly reacts with 5'- phosphate group of adenosine monophosphate to form isopentyl adenine. Examples: A. tumefaciens, Azotobacter chroococcum, Taphrina cerasi, etc.
Cytokinins are extracted from culture broth by using the following simple procedure:
The culture broth is centrifuged to separate the liquid portion from the culture.
The liquid is treated with ammonium hydroxide solution which dissolves cytokinins. This solution is allowed to pass through a DOWEX 50 (H+) column. Owing to the high solubility of cytokinin, it easily drains off from the DOWEX 50 (H+) column.
This solution is again mixed with n- butanol in a separating funnel. The cytokinin dissolves in n- butanol and forms a separate layer which is then separated from the separating funnel.
The separated solution is allowed to evaporate to get crystals of cytokinins. The individual cytokinines are then separated from each other using chromatographic methods. Solvents such as butanol- ammonium hydroxide - water (3:1:1) and acetate- butanol- water (4:1:2) are used for this chromatographic separation.
The extracted cytokinin is then subjected to bioassay.
Miller (1963) has developed a bioassay technique to assess the presence of cytokinin a solution. The different steps of cytokinin bioassay test are summarized below:
The seeds of soyabeans are surface - sterilized properly by using disinfectant. The surface sterilized seeds are then allowed to germinate under aseptic conditions.
After germination the cotyledons of the seedlings are excised and cut into small pieces of 4*4*2 mm size.
The cotyledonary segments are aseptically inoculated into flask containing a semi-solid medium without cytokinin. Similarly, the cotyledonary segments are also inoculated into another flask containing the semi- solid medium with the test solution. Both cultures are incubated at 25-17ºC until they produce calluses. Generally cotyledons take 3 weeks to produce callus tissues.
After 3 weeks the calluses in each flask are weighed properly by using a sensitive balance.
If the weight of the segments taken from the test solution exceeds the weight of the segments taken from the other flask, it indicated that the test solution contains cytokinin- like compounds. If it does not happen, than the test solution is devoid of cytokinins.
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