Blog: The Wheat Sheaf
Microspore Extraction and DNA Isolation
Extracting plant microspores tends to be the first step into manipulating any of the cereal crops. Microspores are immature pollen cells that are capable of forming gametophytes, which will develop sperm cells (the male gametes) to fertilize an egg.
The first step to isolating microspores is to identify the wheat crop you want to work with. Once identified, wheat plants are grown until they start producing tillers. Tillers are collected, spikes are separated and the awns are cut off to allow for easier handling. Next, use sterile forceps to remove each of the florets into a cold container for storage. After filling to the top with solvent, the florets will be blended together to release microspores. The blended contents will be subjected to a series of filtrations to collect the liquid flow through, which contains the single celled microspores. The liquid compartment will undergo centrifugation to further isolate microspore cells from all the other cells and debris.
Centrifugation is the process of sedimentation of a heterogeneous mixture using centripetal force; this is usually done in a machine called centrifuge. This step will allow single cell microspores to settle and form a pellet on the bottom of the solution. The fluid will be drained, and the pellet will be re-suspended with a series of wash solutions. In order to only collect the live microspore cells, a small volume of maltose solution will be added to the suspension, mixed and topped off with wash solution. The interphase between maltose and wash solutions will accumulate all the live microspore cells, leaving all the unwanted dead cells in the bottom layer. Finally, a pipette will be utilized to completely extract the maltose interphase layer. The concentration of the microspore culture can be quantified by using a cell counting device, for example a hemocytometer, and diluent can be added to the culture to adjust to the ideal concentration.
This microspore extraction protocol is a very useful tool to do further advanced methodologies, such as embryogenesis, doubled haploid technique, genetics and genomic work, and many other molecular studies.
DNA Isolation and How to Extract your Target Gene:
Traditionally, isolating DNA from a cell involved a fairly advanced protocol with many different steps. Now, isolating DNA can be done by using a small kit with solutions already pre-prepared for you. It’s so simple that everyone can isolate DNA now!
So now that you have the DNA, how do you extract your target gene? There are many ways to do this, but one of the simpler methods involve utilizing restriction enzymes that will cut up the entire genomic DNA, but will not cut through your target gene. There are many different restriction enzymes in the market, and usually more than one restriction enzyme will be used so that the DNA ends will not stick back together.
The next step will be to prepare the agarose gel apparatus. Agarose is a polymer that is mainly obtained from seaweed. This gel is very commonly used in molecular studies to separate molecules, especially DNA. Loading dye will be added to the chopped up DNA for visibility (because DNA is transparent to our eyes), and the entire solution will be loaded onto the agarose gel. Agarose gel electrophoresis is the application of voltage into the gel. DNA is negative charge, so the application of voltage will drive individual DNA molecules toward the positive side of the apparatus. The shorter the DNA is, the faster it’ll migrate through the gel.
Ethidium bromide is usually added to the gel prior to applying voltage. Ethidium bromide is a potential carcinogen as it is an intercalating agent, meaning it can enter and attach to DNA. This agent is usually used as it gives a fluorescent tag to DNA under UV light, making the DNA bands visible in the gel. There are other alternatives to ethidium bromide, such as SYBR Green and GelRed. Usually, the technician working on the gene will know which DNA fragment contains the target gene and where it is located on the gel after electrophoresis. A short knife will be used to cut the target gene out from the gel, and will be placed into a gel extraction kit to remove any gel fragments that remained during the cut. Finally, you can sequence the extracted gene to ensure you have obtained the correct DNA.