SafteyGibberellic acid may cause irritation of skin and eyes. Wear eye protection and avoid skin contact. Wash your hands after use. 3% sodium hypochlorite solution (bleach) is an irritant. Wear eye protection. Use in a well-ventilated space. The incubated agar plates are a potential biohazard, though a hazard is unlikely with _ starch agar. Practise good aseptic technique and do not completely remove lids. Wash hands after handling. Sharp scalpels may cause cuts. Hold seeds in forceps when cutting. Equipment
Independant VariableConcentration of gibberellin in ugdm -3 Dependant VariableDiameter of clear area around cereal grain where there is no starch Control Variables
ProcedureA cereal grain contains a store of starch within the endosperm. During germination the starch must be made soluble so that it can be transported to the embryo to support the growth of the seedling. The embryo is much smaller than the endosperm and is situated at the more pointed end of the grain. The developing embryo releases gibberellins that act on a layer of cells on the outside of the endosperm, stimulating these cells to release the starch-digesting enzyme amylase. In this activity you will remove the embryo and investigate the effect of different concentrations of gibberellin on the production of amylase. The production of amylase will be assessed by using a starch agar assay. Cereal grains that have had the embryo removed are first soaked in gibberellic acid, then placed onto the starch agar plates and incubated. The agar plate is then flooded with iodine solution, which stains starch blue-black. The areas where starch has been digested will not stain. The size of the clear area around a cereal grain indicates the amount of amylase produced by the seed. Planning The basic procedure is outlined below but you will need to produce a plan before you start. This should provide detail and justify your decisions on how you will do the following. Set the levels of the independent variable (gibberellin concentration). We will use a range of 6 different concentrations from 0 ugdm-3 to 500 ugdm-3. We are using intervals of 100 umgdm-3. 0cugdm-3 is our control variable to allow us yo compare what effect having no gibberellin has on an endosperm Measure the dependent variable (size of the clear area). Finally we need to ensure that we control any other factors that we are not measuring. This means that gibberellin concentration is the only variable that we are measuring making our experiment valid. It is important that a good sterile technique is used throughout this investigation to prevent the growth of microorganisms that could present a biohazard. Day 1 1. Make up your gibberellin solutions as planned. Only small volumes (a few cm3) of each are required. Place each solution in a small labelled sample bottle. 2. Collect the number of seeds that you require and pull any husks off the grains so the shape can be seen clearly. 3. Cut each seed across the line X-Y (see fig A) so that one half contains the embryo and the other the endosperm. Keep the two halves separate and discard the seed halves that contain the embryo. 4. Sterilise the remaining endosperm halves of the seeds by placing them in 3% sodium hypochlorite solution for 5 minutes. 5. Wash the seeds thoroughly, but quickly. through have changes of sterile water, draining carefully through muslin each time, until there is no smell of chlorine. Drain fully. Secondary ResearchFrom looking at secondary research I have learnt that gibberellin acts as a transcription factor in the cereal grains. The gibberellin binds to the DNA at the promoter sequences at the points where it transcribes for the code that produces the proteins that make up the enzyme amylase. The research was carrried out be Sang-Choo Lee and his team in the University of Seoul. http://www.sciencedirect.com/science/article/pii/S0176161717301062
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