Discovering The Genetic Potential Of Your Plants

Molecular Plant Biology

BY DR CALLIE SEAMAN

Back when I was doing my undergraduate degree, molecular biology would fry my brain. I would dread the three-hour lectures every Thursday morning. I didn’t realize how useful that course was until recently. Molecular biology is described as the activities and interactions that occur between biomolecules such as DNA, RNA and proteins within cells and within the whole organism. The synthesis and metabolism of these biomolecules have a lot to do with plant genetics. Please refer to the glossary at the end of this article to help with some of the terminologies.

Studying this branch of biology tells us a lot about the plants we are growing. It helps determine the chemotype (the chemicals and secondary metabolites it will produce), disease susceptibility, drought resistances, phenotype (physical characteristics which are determined by genetics, including plant size, leaf shape, etc.,), sex, heritage or lineage, and diseases present. With recent advances, molecular biology has also been able to quantify microbial contamination such as Aspergillus and Fusarium within a sample.

How Is This Possible?

We have all watched CSI Miami or NCIS on TV; I hate to burst your bubble, but labs are NOT mood-lit with blue LEDs, and the analysis does not take ten minutes. So much more preparation goes into determining who killed Col. Mustard in the library! One of the critical tools in the chest of a molecular biologist is the polymerase chain reaction, also known as PCR. This technique amplifies a specific section of DNA, replicating it many times so we can more easily identify it.

We have all watched CSI Miami or NCIS on TV; I hate to burst your bubble, but labs are NOT moodlit with blue LEDs, and the analysis does not take ten minutes

PCR: polymerase chain reaction

credit: https://en.wikipedia.org/wiki/Polymerase_chain_reaction

[PCR] can save time by identifying the sex of a plant with in a week of germination, which in turn helps to reduce costs on power, space, and time

How Does It Work?

PCR involves several steps and heating cycles; first, there’s the extraction of the DNA before splitting it open (Denaturation). Then, specific primers are attached to the known segments of DNA (primer annealing). Finally, the amplification of the target DNA (polymerization) takes place. There is always the risk of contamination with the samples, and false-positive or negatives are possible. There are lovely kits called thermocyclers that are pre-programed with the cycles of heating and cooling and make life a little easier during this process.

When examining genetics, the DNA must be first extracted from the cells using a mixture of ionic salts (sodium chloride) and buffering salts (Tri HCl) which are subjected to a heating cycle. This process breaks open the cells, releasing the DNA. After this, the annealing process begins with specific oligonucleotide primers attached to the target DNA at a lower temperature. The temperature is increased again to optimize the polymerization process and amplify the DNA. An indicator solution is then used to provide a positive or negative result.

How Do We Apply This To Real Life?

If, for example, we are looking to see if the plant is infected with botrytis, the genes which are expressed by the plant are particular and would stick to the matching primer we add. This Pritt-Sticked piece of DNA is then replicated many times during a final heating cycle. We now have a soup of one particular strand of DNA, which allows for straightforward detection. If these are not present, then the DNA will not be replicated, and an indicator solution would not react. Further quantification can be done of the sample using qPCR, which utilizes a fluorescent probe/marker that varies in intensity with increased concentration of the DNA of interest and is measured by a qPCR instrument. Real-time PCR (RTPCR), on the other hand, tells us if the gene of interest is present or not.

Why use this qPCR technique over traditional microbiology culture plating methods? This is a relatively simple technique to perform in comparison to practices such as microarray or culture plating. When it comes to the genetics of the microbes such as e.coli, salmonella, or powdery mildew, specific species can be identified rather than generalized identification. PCR is fast, accurate, and is also more compatible with a variety of different matrices (the sample you are testing). It gives the grower an advantage, almost allowing them to look into the future and see what the plant’s genetic potential is. It can save time by identifying the sex of a plant within a week of germination, which in turn helps to reduce costs on power, space, and time. It also allows a grower to adapt their growing techniques to help prevent disease outbreaks, for example, by applying extra silicon products to the feed regime if the sample yielded a positive result for botrytis.

Glossary

• Bases – The most basic building blocks of DNA made up of Guanine, Cytosine, Adenine and Thymine/Uracil, pairing respectively (G-C and A-T)

• Nucleotides – This is what forms when the bases fuse with sugar to form the most basic structure of DNA formed by the building blocks.

• Oligonucleotide – a small number of nucleotides joined together.

• DNA – deoxyribonucleic acid and is the chemical code that determines our characteristics from previous generations. It is made up of base pairs, Guanine, Cytosine, Adenine and Thymine/Uracil (G-C and A-T).

• Gene – a gene is made up of sequences of DNA and can vary from a few hundred strands of DNA to well into the millions. These are what we inherit from our parents.

• Primer – This provides the starting point for DNA replication to take place and is used in analytical techniques to help replicate DNA.

• Chromosome – is a molecule of DNA that contains all or part of the genetic info of the organism. They also contain proteins that aid with the structure, and the integrity of the molecule. These contain genes and are specific to each organism. Chromosomes dictate sex.

References

• Charlesworth, D. Plant sex determination and sex chromosomes. Heredity 88, 94–101 (2002) doi:10.1038/sj.hdy.6800016

• Leister, Dario, Agim Ballvora, Francesco Salamini, and Christiane Gebhardt. “A PCR–based approach for isolating pathogen resistance genes from potato with potential for wide application in plants.” Nature genetics 14, no. 4 (1996): 421.

• Ingham, David J., Sandra Beer, Stephanie Money, and Geneviève Hansen. “Quantitative real-time PCR assay for determining transgene copy number in transformed plants.” Biotechniques 31, no. 1 (2001): 132-140.

• Turner, Phil, Alexander McLennan, Andy Bates, and Michael White. BIOS Instant Notes in Molecular Biology. Taylor & Francis, 2007.

BIO Dr. Callie Seaman is a plant obsessed Formulation Chemist at AquaLabs – the company behind SHOGUN Fertilizers and the Silver Bullet plant health range. She has been in the hydro industry for 15 years in research development and manufacturing and had previously worked on the VitaLink range. She has a PhD in fertilizer chemistry and a BSc (HONS) in Biomedical sciences and loves nothing more than applying this knowledge to pushing the boundaries of nutrient performance.