Other Tissue Culture Topics 2009

Other Tissue Culture Topics 2009 - Other Plant Tissue...

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Other Plant Tissue Topics
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Somaclonal Variation Somaclonal variation is a general phenomenon of all plant regeneration systems that involve a callus phase There are two general types: Heritable, genetic changes (alter the DNA) Stable, but non-heritable changes (alter gene expression, epigenetic) With or without mutagen
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Somaclonal/Mutation Breeding Advantages: Screen very high populations (cell based) Can apply selection to single cells Disadvantages: Many mutations are non-heritable Requires dominant mutation (or double recessive mutation); most mutations are recessive Can avoid this constraint by not applying selection pressure in culture, but you lose the advantage of high through-put screening –have to grow out all regenerated plants, produce seed, and evaluate the M 2
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Somaclonal Breeding Procedures Use plant cultures as starting material Idea is to target single cells in multi-cellular culture Usually suspension culture, but callus culture can work Optional: apply physical or chemical mutagen Apply selection pressure to culture? Target: very high kill rate, you want very few cells to survive, so long as selection is effective Regenerate whole plants from surviving cells
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Requirements for Somaclonal Breeding Effective screening procedure Most mutations are deleterious » With fruit fly, the ratio is ~800:1 deleterious to beneficial Most mutations are recessive » Must screen M 2 or later generations » Consider using heterozygous plants? » Haploid plants seem a reasonable alternative if possible Very large populations are required to identify desired mutation: » Can you afford to identify marginal traits with replicates & statistics? Estimate: ~10,000 plants for single gene mutant Clear Objective
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Introduction into suspension + Plate out Sieve out lumps 1 2 Initial high density Subculture and sieving
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Synchronization Cold treatment: 4 o C Starvation: deprivation of an essential growth compound, e.g. N →accumulation in G1 Use of DNA synthesis inhibitors: thymidine, 5-fluorodeoxyuridine, hydroxyurea Colchicine method: arresting the cells in metaphase stage, measured in terms of mitotic index (% cells in the mitotic phase)
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Select at the level of the intact plant Select in culture single cell is selection unit possible to plate up to 1,000,000 cells on a Petri- dish. progressive selection over a number of phases
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This note was uploaded on 07/23/2011 for the course HOS 6737c taught by Professor Moore during the Spring '09 term at University of Florida.

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Other Tissue Culture Topics 2009 - Other Plant Tissue...

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